Anti-CCR5 antibody

ABSTRACT

The invention is directed an anti-CCR5 antibody which comprises (i) two light chains, each light chain comprising the expression product of a plasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii), two heavy chains, each heavy chain comprising an expression product of either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099) or a fragment thereof which binds to CCR5 on the surface of a human cell.

This application is a continuation-in-part of and claims the priority ofU.S. Provisional Application No. 60/358,886, filed Feb. 22, 2002, thecontents of which are hereby incorporated by reference into thisapplication.

Throughout this application, various publications are referenced byArabic numerals. Full citations for these publications may be found atthe end of the specification immediately preceding the claims. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the art to which thisinvention pertains.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) induces viral-to-cellmembrane fusion to gain entry into target cells (8, 15, 66). The firsthigh-affinity interaction between the virion and the cell surface is thebinding of the viral surface glycoprotein gp120 to the CD4 antigen (13,30, 41, 42). This in turn induces conformational changes in gp120, whichenable it to interact with one of several chemokine receptors (4, 5, 21,36). The CC-chemokine receptor CCR5 is the major co-receptor formacrophage-tropic (R5) strains, and plays a crucial role in the sexualtransmission of HIV-1 (4, 5, 21, 36). T cell line-tropic (X4) virusesuse CXCR4 to enter target cells, and usually, but not always, emergelate in disease progression or as a consequence of virus propagation intissue culture (4, 5, 21, 36). Some primary HIV-1 isolates aredual-tropic (R5×4) since they can use both co-receptors, though notalways with the same efficiency (11, 57). Mutagenesis studies coupledwith the resolution of the gp120 core crystal structure demonstratedthat the co-receptor-binding site on gp120 comprises several conservedresidues (32, 53, 65).

It has been demonstrated that tyrosines and negatively charged residuesin the amino-terminal domain (Nt) of CCR5 are essential for gp120binding to the co-receptor, and for HIV-1 fusion and entry (6, 18, 20,22, 28, 31, 52, 54). Residues in the extracellular loops (ECL) 1-3 ofCCR5 were dispensable for co-receptor function, yet the CCR5inter-domain configuration had to be maintained for optimal viral fusionand entry (24). This led to the conclusion either that gp120 formsinteractions with a diffuse surface on the ECLs, or that the Nt ismaintained in a functional conformation by bonds with residues in theECLs. Studies with chimeric co-receptors and anti-CCR5 monoclonalantibodies have also shown the importance of the extracellular loops forviral entry (5, 54, 64).

Molecules that specifically bind to CCR5 and CXCR4 and blockinteractions with their ligands are a powerful tool to further probe thestructure/function relationships of the co-receptors. Characterizingsuch compounds could also assist in designing effective therapeuticagents that target co-receptor-mediated steps of viral entry. Inhibitorsof CCR5 or CXCR4 co-receptor function identified to date are diverse innature and include small molecules, peptides, chemokines and theirderivatives, and monoclonal antibodies (mAbs). The mechanisms of actionof the small molecules that block entry by interfering with CXCR4co-receptor function are not well understood (17, 49, 55, 68). One suchinhibitor, the anionic small molecule AMD3100, depends on residues inECL2 and the fourth trans-membrane (TM) domain of CXCR4 to inhibit viralentry, but it is not clear whether it does so by disrupting gp120binding to CXCR4 or post-binding steps leading to membrane fusion (16,34, 55). To date, no small molecules have been reported thatspecifically block CCR5-mediated HIV-1 entry. Inhibition of HIV-1 entryby chemokines is mediated by at least two distinct mechanisms: blockageof the gp120/co-receptor interaction and internalization of thechemokine/receptor complex (3, 26, 59, 63). The variant AOP-RANTES alsoinhibits recycling of CCR5 to the cell surface (40, 56). Variants suchas RANTES 9-68 and Met-RANTES only prevent the gp120/CCR5 interactionand do not down-regulate CCR5 (67). SDF-1 variants presumably actthrough a similar mechanism to block viral entry mediated by CXCR4 (12,27, 39). Only one anti-CXCR4 mAb, 12G5, has been characterized for itsanti-viral properties. The efficiency of 12G5 inhibition of viral entryhas been reported to be both cell- and isolate-dependent (43, 58). ThismAb binds to the ECL2 of CXCR4, but the mechanism by which it inhibitsentry is unknown (7). Few of the anti-CCR5 mAbs characterized to dateefficiently prevent HIV-1 entry (28, 64). Interestingly, mAbs whoseepitopes lie in the Nt domain of CCR5, which contains the gp120-bindingsite, inhibit viral fusion and entry less efficiently than mAb 2D7,whose epitope lies in ECL2. 2D7 also antagonizes CC-chemokine activity(64).

A panel of six murine mAbs, designated PA8, PA9, PA10, PA11, PA12 andPA14 have been isolated and characterized. All six mAbs specificallybound to CCR5⁺ cells but with different efficiencies that were celltype-dependent. Epitope mapping studies identified the residues that areimportant for mAb binding and also revealed information about thefolding and interactions of the CCR5 extracellular domains. All mAbsinhibited HIV-1 fusion and entry, but there was no correlation betweenthe ability of a mAb to inhibit fusion and entry and its ability toinhibit binding of gp120/sCD4 to CCR5⁺ cells.

SUMMARY OF THE INVENTION

This invention provides an anti-CCR5 antibody which comprises (i) twolight chains, each light chain comprising the expression product of aplasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097),and (ii) two heavy chains, each heavy chain comprising the expressionproduct of either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCCDeposit Designation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), or a fragment of suchantibody, which binds to CCR5 on the surface of a human cell.

This invention also provides an anti-CCR5 antibody comprising two lightchains, each chain comprising consecutive amino acids, the amino acidsequence of which is set forth in SEQ ID NO:6, and two heavy chains,each heavy chain comprising consecutive amino acids, the amino acidsequence of which is set forth in SEQ ID NO:9.

This invention also provides an anti-CCR5 antibody comprising two lightchains, each chain comprising consecutive amino acids, the amino acidsequence of which is set forth in SEQ ID NO:6, and two heavy chains,each heavy chain comprising consecutive amino acids, the amino acidsequence of which is set forth in SEQ ID NO:12.

This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO:6. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO:5.

This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO:9. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO:8.

This invention also provides an isolated nucleic acid encoding apolypeptide comprising consecutive amino acids, the amino acid sequenceof which is set forth in SEQ ID NO:12. In the subject embodiment, thenucleic acid comprises the sequence set forth in SEQ ID NO:11.

This invention also provides a composition comprising at least oneanti-CCR5 antibody, or a fragment thereof, as described above, togetherwith a carrier.

This invention also provides a composition comprising the anti-CCR5antibody, or a fragment thereof, having attached thereto a material suchas a radioisotope, a toxin, polyethylene glycol, a cytotoxic agentand/or a detectable label.

This invention also provides a method of inhibiting infection of a CD4+cell which comprises contacting the CD4+ cell with an antibody whichcomprises (i) two light chains, each light chain comprising theexpression product of a plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097), and (ii) two heavy chains, each heavy chaincomprising the expression product of either a plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099), or a fragment of such antibody which binds to CCR5 on thesurface of a CD4+ cell, in an amount and under conditions such thatfusion of HIV-1 or an HIV-1-infected cell to the CD4+ cell is inhibited,thereby inhibiting HIV-1 infection of the CD4+ cell.

This invention also provides a method of treating a subject afflictedwith HIV-1 which comprises administering to the subject an effectiveHIV-1 treating dosage of an anti-CCR5 antibody comprising (i) two lightchains, each light chain comprising the expression product of a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii)two heavy chains, each heavy chain comprising the expression product ofeither a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), or a fragment of suchantibody, which binds to CCR5 on the surface of a human cell, underconditions effective to treat the HIV-1-infected subject.

This invention also provides a method of preventing a subject fromcontracting an HIV-1 infection which comprises administering to thesubject an effective HIV-1 infection-preventing dosage amount of ananti-CCR5 antibody comprising (i) two light chains, each light chaincomprising the expression product of a plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavychains, each heavy chain comprising the expression product of either aplasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCCDeposit Designation PTA-4099), or a fragment of such antibody, whichbinds to CCR5 on the surface of a human cell, under conditions effectiveto prevent the HIV-1, infection in the subject.

This invention also provides an anti-CCR5 antibody conjugate comprisingan anti-CCR5 antibody which comprises (i) two light chains, each lightchain comprising the expression product of a plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii) two heavychains, each heavy chain comprising the expression product of either aplasmid designated pVg1:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098) or a plasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCCDeposit Designation PTA-4099), or a fragment of such antibody whichbinds to CCR5 on the surface of a human cell, conjugated to at least onepolymer.

This invention also provides a method of inhibiting infection of a CCR5+cell by HIV-1 comprising administering to a subject at risk of HIV-1infection the above-described conjugate in an amount and underconditions effective to inhibit infection of CCR5+ cells of the subjectby HIV-1.

This invention also provides a method of treating an HIV-1 infection ina subject comprising administering the above-described conjugate to anHIV-1-infected subject in an amount and under conditions effective totreat the subject's HIV-1 infection.

This invention also provides a transformed host cell comprising at leasttwo vectors, at least one vector comprising a nucleic acid sequenceencoding heavy chains of an anti-CCR5 antibody, and at least one vectorcomprising a nucleic acid sequence encoding light chains of theanti-CCR5 antibody, wherein the anti-CCR5 antibody comprises two heavychains having the amino acid sequence set forth in SEQ ID NO:9, and twolight chains having the amino acid sequence set forth in SEQ ID NO:6.

This invention also provides a transformed host cell comprising at leasttwo vectors, at least one vector comprising a nucleic acid sequenceencoding heavy chains of an anti-CCR5 antibody, and at least one vectorcomprising a nucleic acid sequence encoding light chains of theanti-CCR5 antibody, wherein the anti-CCR5 antibody comprises two heavychains having the amino acid sequence set forth in SEQ ID NO:12 and twolight chains having the amino acid sequence set forth in SEQ ID NO:6.

This invention also provides a vector comprising a nucleic acid sequenceencoding a heavy chain of an anti-CCR5 antibody, wherein the heavy chaincomprises the amino acid sequence set forth in SEQ ID NO:9.

This invention also provides a vector comprising a nucleic acid sequenceencoding a heavy chain of an anti-CCR5 antibody, wherein the heavy chaincomprises the amino acid sequence set forth in SEQ ID NO:12.

This invention also provides a process for producing an anti-CCR5antibody which comprises culturing a host cell containing therein (i) aplasmid designated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097),and (ii) either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099) under conditionspermitting the production of an antibody comprising two light chainsencoded by the plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation

PTA-4097) and two heavy chains encoded either by the plasmid designatedpVg1:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or by theplasmid designated pVg1:HuPRO140 (mut B+D+I)-VH (ATCC DepositDesignation PTA-4099), so as to thereby produce an anti-CCR5 antibody.

This invention also provides a process for producing an anti-CCR5antibody which comprises a) transforming a host cell with (i) a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097) and (ii)either a plasmid designated pVg1:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg1:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099), and b) culturing thetransformed host cell under conditions permitting production of anantibody comprising two light chains encoded by the plasmid designatedpVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097) and two heavy chainsencoded either by the plasmid designated pVg1:HuPRO140 HG2-VH (ATCCDeposit Designation PTA-4098) or by the plasmid designated pVglHuPRO140(mut B+D+I)-VH (ATCC Deposit Designation PTA-4099), so as to therebyproduce an anti-CCR5 antibody.

This invention also provides a kit for use in a process of producing ananti-CCR5 antibody. The kit comprises a) a vector comprising a nucleicacid sequence encoding a light chain of an anti-CCR5 antibody, whereinthe light chain comprises the amino acid sequence set forth in SEQ IDNO:6, and b) a vector comprising a nucleic acid sequence encoding aheavy chain of an anti-CCR5 antibody, wherein the heavy chain comprisesthe amino acid sequence set forth in SEQ ID NO:9, or a vector comprisinga nucleic acid sequence encoding a heavy chain of an anti-CCR5 antibody,wherein the heavy chain comprises the amino acid sequence set forth inSEQ ID NO:12.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

Binding of Anti-CCR5 Monoclonal Antibodies to CCR5⁺ Cells:

Flow cytometry was used to detect CCR5 protein expression on the surfaceof L1.2-CCR5⁺ cells and freshly isolated, PHA/IL-2-stimulated PBMC.Cells were incubated with saturating concentrations of each mAb, whichwere detected with a PE-labeled anti-mouse IgG reporter antibody.Results from a representative experiment are shown. Results for each mAbare expressed both in mean fluorescence intensities (m.f.i.) and in %gated cells. Since PA8-PA12 and PA14 are all of the IgG1 subclass, theirm.f.i. are directly comparable. 2D7 is an IgG2a.

FIG. 2:

CI Values for Different Combinations of mAbs and Viral Inhibitors:

Experiments like those described in the legend of FIG. 7 were performedfor different combinations of viral entry inhibitors. Anti-CCR5 mAbswere tested in combination with each other, CC-chemokines, and CD4-IgG2,which inhibits HIV-1 attachment to target cells. The PA11 and PA12concentration range was 0-250 μg/ml; the 2D7 and PA14 concentrationrange was 0-25 μg/ml; the RANTES concentration range was 0-250 ng/ml;the CD4-IgG2 concentration range was 0-25 μg/ml. The concentrations ofsingle-agents or their mixtures required to produce 50% and 90%inhibition of fusion or entry were quantitatively compared in a termknown as the Combination Index (CI).

FIG. 3:

IC₅₀ Values for Inhibition of Cell-Cell Fusion, Viral Entry andgp120/sCD4 Binding by Anti-CCR5 mAbs:

For comparative purposes we have summarized the IC₅₀ values obtained inthe different assays that the anti-CCR5 mAbs were tested in. IC₅₀ valueswere only calculated for mAbs that could inhibit >90% of fusion, entryor binding.

FIG. 4:

Epitope Mapping of Anti-CCR5 mAbs:

A two color staining protocol was used to assess binding of mAbs tomutant CCR5 proteins, tagged at the C-terminus with the HA peptide. HeLacells expressing CCR5 point mutants were incubated with saturatingconcentrations of each mAb followed by detection with a PE-labeledanti-mouse IgG. Cell surface co-receptor expression was measured bydouble-staining of the cells with a FITC labeled anti-HA mAb. The fourgrids correspond to the four extracellular domains of CCR5. The firstrow of every grid indicates the amino acid sequence of the correspondingCCR5 extracellular domain (SEQ ID NOS: 1-4). Binding of anti-CCR5 mAbsto the alanine mutant of each residue is expressed as a percentage ofbinding to wild-type CCR5, as described in Materials and Methods.

FIG. 5:

Inhibition of Calcium Mobilization into CCR5 Cells by Anti-CCR5 mAbs:

L1.2-CCR5⁺ cells were loaded with Indo-1AM and stimulated sequentiallywith an anti-CCR5 mAb or PBS, followed with RANTES (a). Fluorescencechanges were measured with a spectrofluorometer and the tracings arefrom a representative experiment. Calcium flux inhibition by PA14 and2D7 was tested for a wide range of mAb concentrations (b). Results areplotted as % inhibition of calcium influx=[1−(relative fluorescence inthe presence of mAb relative fluorescence in the absence of mAb)]×100%,and are means of values from three independent experiments.

FIG. 6:

Inhibition of CCR5 co-receptor function by anti-CCR5 mAbs:

Inhibition of cell-cell fusion by anti-CCR5 mAbs was tested in the RETassay (a). 0-250 μg/ml of PA8-PA12, or 0-25 μg/ml of PA14 or 2D7, wereadded to a mix of HeLa-Env_(JR-FL) ⁺ and PM1 cells, labeled with F18 andR18 respectively. Fluorescence RET was measured after 4 h of incubation.Results are mean values from three independent experiments and areexpressed as % inhibition of fusion=[1−(% RET in the presence of mAb÷%RET in the absence of mAb)]×100%. Inhibition of HIV-1 entry by anti-CCR5mAbs was tested in a single round of replication luciferase based entryassay (b). U87-CD4⁺CCR5⁺ cells were infected with NLluc⁺env⁻ reportervirus carrying the JR-FL envelope in the presence of 0-250 μg/ml ofPA8-PA12, or 0-25 μg/ml PA14 or 2D7. Luciferase activity (relative lightunits, r.l.u.) was measured in cell lysates 72 h post-infection. Resultsare from a representative experiment and are expressed as % inhibitionof entry=[1−(r.l.u. in the presence of mAb÷r.l.u. in the absence ofmAb)]×100%. Binding of biotinylated [b] gp120, sCD4 and b-gp120-CD4complexes to L1.2-CCR5⁺ cells (c). Strong binding is observed when gp120derived from the R5 virus HIV-1_(JR-FL) is complexed with an equimolaramount of sCD4. No binding is observed in the absence of sCD4 or forgp120 derived from the X4 virus HIV-1_(LAI). Background binding toCCR5-L1.2 cells has been subtracted from all curves. Inhibition ofgp120/sCD4 binding to L1.2-CCR5⁺ cells was tested in the presence ofvarying concentrations of each antibody (d). Cells were pre-incubated in96-well plates with an anti-CCR5 mAb followed by an incubation with asaturating concentration of biotinylated gp120/sCD4. Finally, binding ofPE-labeled streptavidin to cells was measured using a fluorescence platereader. Results are from a representative experiment and are expressedas % inhibition of gp120/sCD4 binding=[1−(m.f.i. in the presence ofmAb÷m.f.i. in the absence of mAb)]×100%.

FIG. 7:

Synergistic Inhibition of Cell-Cell Fusion by PA12 and 2D7:

Dose-response curves were obtained for the mAbs used individually and incombination. 0-50 μg/ml of PA12, 0-25 μg/ml 2D7, or a combination of thetwo in a 2:1 ratio, were added to a mix of HeLa-Env_(JR-FL) ⁺ and PM1cells, labeled with R18 and F18 respectively. Fluorescence RET wasmeasured after 4 hours of incubation. Results are expressed as %inhibition of fusion and are the means of values from three independentexperiments. Data were analyzed using the median effect principle, whichcan be written

f=1/[1+(K/c)·^(m)]  (1)

where f is the fraction affected/inhibited, c is concentration, K is theconcentration of agent required to produce the median effect, and m isan empirical coefficient describing the shape of the dose-responsecurve. Equation (1) is a generalized form of the equations describingMichaelis-Menton enzyme kinetics, Langmuir adsorption isotherms, andHenderson-Hasselbalch ionization equilibria, for which m=1. In thepresent case, K is equal to the IC₅₀ value. K and m were determined bycurve-fitting the dose-response curves and Equation (1) was rearrangedto allow calculation of c for a given f. The best-fit parameters for Kand c are 8.8 μg/ml and 0.54 for PA12, 0.36 μg/ml and 0.68 for 2D7, and0.11 μg/ml and 1.1 for their combination. These curves are plotted andindicate a reasonable goodness-of-fit between experiment and theory.

FIG. 8:

This figure shows the amino acid sequence of the light chain variableregion of a humanized version of mouse anti-CCR5 antibody PA14 (SEQ IDNO: 6) and the nucleic acid sequence encoding the same (SEQ ID NO: 5),in accordance with the invention. SEQ ID NO: 7 identifies the region ofSEQ ID NO: 5 which codes for the amino acid sequence set forth in SEQ IDNO:6. This light chain variable region is present in the antibodiesdesignated herein as PRO 140 #1 and #2. The complementarity-determiningregions (“CDRs”) are underlined.

FIG. 9:

This figure shows the amino acid sequence of a first heavy chainvariable region of a humanized version of mouse anti-CCR5 antibody PA14(SEQ ID NO:9), and the nucleic acid sequence encoding the same (SEQ IDNO:8), in accordance with the invention. SEQ ID NO:10 identifies theregion of SEQ ID NO:8 that codes for the amino acid sequence set forthin SEQ ID NO:9. This heavy chain variable region is present in theantibody designated herein as PRO 140 #2. The CDRs are underlined.

FIG. 10:

This figure shows the amino acid sequence of a second heavy chainvariable region of a humanized version of mouse humanized anti-CCR5antibody PA14 (SEQ ID NO:12) and the nucleic acid sequence encoding thesame (SEQ ID NO:11) in accordance with the invention. SEQ ID NO:13identifies the region of SEQ ID NO:11 that codes for the amino acidsequence set forth in SEQ ID NO:12. This heavy chain variable region ispresent in the antibody designated herein as PRO 140 #1. The CDRs areunderlined.

FIG. 11:

Single-Dose of Humanized CCR5 Antibody Potently Reduces Viral Loads InVivo:

SCID mice were reconstituted with normal human PBMC and infected withHIV-1_(JR-CSF). When a viral steady state was reached, the animals weretreated with a single 1 milligram i.p. dose of humanized CCR5 antibody(PRO 140) or isotype control antibody and monitored for plasma HIV RNA(Roche Amplicor Assay).

FIG. 12:

Sustained Reduction in Viral Load:

SCID mice were reconstituted with normal human PBMC and infected withHIV-1_(JR-CSF). When a viral steady state was reached, the animals weretreated i.p. with 0.1 mg doses of humanized CCR5 antibody (PRO140) everythree days and monitored for plasma HIV RNA (Roche Amplicor Assay).

FIG. 13:

Demonstrates that there was no depletion of lymphocytes with the use ofthe CCR5 antibody (PRO 140) prepared in accordance with the invention.

FIG. 14:

Humanized CCR5 Antibody (PRO140) Potently Blocks CCR5-Mediated HIV-1Cell-Cell Fusion.

Murine CCR5 antibody was humanized using the method ofcomplementarity-determining region (CDR) grafting and frameworksubstitutions. Humanized CCR5 antibodies (PRO 140 #1 and PRO 140 #2)were expressed in Sp2/0 cells, purified by protein A chromatography andtested for the ability to block replication of HIV-1_(JR-FL)env-mediated membrane fusion as described (Litwin, et al., J, Virol.,70:6437, 1996).

FIG. 15:

Humanized CCR5 Antibody (PRO 140) Mediates Potent, Subtype-IndependentInhibition of HIV-1.

CCR5 Antibodies (Pro 140 #1 and #2) according to the invention weretested for the ability to block replication of wild-type HIV-1 inperipheral blood mononuclear cells (PBMCs) as described (Trkola et al.,J. Virol., 72:396, 1998). The extent of viral replication was measuredby assaying the p24 antigen content of 7-day PBMC culture supernatants.

FIG. 16:

This figure provides a map of plasmid pVK-HuPRO140 encoding the lightplasmid chain variable region shown in FIG. 8 as well as the human Kappaconstant regions as described in Co et al., J. Immunol., 148:1149, 1992.

FIG. 17:

This figure provides a map of plasmid pVg4-HuPRO140 HG2 encoding theheavy chain variable region shown

in FIG. 9 as well as the human heavy chain constant regions, CH1, hinge,CH2, and CH3, of human IgG4 as described in Co et al, Supra.

FIG. 18:

This figure provides a map of plasmid pVg4-HuPRO140 (mut B+D+I) encodingthe heavy chain variable region shown in FIG. 10 as well as the humanheavy chain constant regions, CH1, hinge, CH2, and CH3, of human IgG4 asdescribed in Co et al, Supra.

FIG. 19

Hu PRO140 Blocks HIV-1 But Not RANTES Signaling

PRO140 antibodies according to the invention were tested for the abilityto block RANTES-induced calcium mobilization in L1.2-CCR5 cells (Olson,et al., J. Virol., 72:396, 1998). This figure shows that a humanizedCCR5 antibody (huPRO140) blocks HIV-1 but not RANTES signaling.

DETAILED DESCRIPTION OF THE INVENTION

The plasmids designated as HuPRO140-VK, HuPRO140 (mut+B+D+I)-VH, andHuPRO140 HG2-VH, which are referred to in FIGS. 16, 18, and 17 aspVK-HuPRO140, pVg4-HuPRO140 (mut B+D+I) and pVg4-HuPRO140 HG2,respectively, were deposited with the American Type Culture Collection,Manassas, Va., U.S.A. 20108 on Feb. 22, 2002, under ATCC Accession Nos.PTA 4097, PTA 4099 and PTA 4098 respectively. These deposits were madepursuant to the provisions of the Budapest Treaty on the internationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure (Budapest Treaty)

This invention provides a composition for inhibiting HIV-1 infectioncomprising at least two compounds in synergistically effective amountsfor inhibiting HIV-1 infection, wherein at least one of the compoundsprevents with the productive interaction between HIV-1 and an HIV-1fusion co-receptor.

As used herein, “composition” means a mixture. The compositions includebut are not limited to those suitable for oral, rectal, intravaginal,topical, nasal, opthalmic, or parenteral administration to a subject. Asused herein, “parenteral” includes but is not limited to subcutaneous,intravenous, intramuscular, or intrasternal injections or infusiontechniques.

As used herein, “HIV-1” means the human immunodeficiency virus type-1.HIV-1 includes but is not limited to extracellular virus particles andthe forms of HIV-1 found in HIV-1 infected cells.

As used herein, “HIV-1 infection” means the introduction of HIV-1genetic information into a target cell, such as by fusion of the targetcell membrane with HIV-1 or an HIV-1 envelope glycoprotein⁺ cell. Thetarget cell may be a bodily cell of a subject. In the preferredembodiment, the target cell is a bodily cell from a human subject.

As used herein, “inhibiting HIV-1 infection” means the reduction of theamount of HIV-1 genetic information introduced into a target cellpopulation as compared to the amount that would be introduced withoutsaid composition.

As used herein, “compound” means a molecular entity, including but notlimited to peptides, polypeptides, and other organic or inorganicmolecules and combinations thereof.

As used herein, “synergistically effective” means that the combinedeffect of the compounds when used in combination is greater than theiradditive effects when used individually.

As used herein, “productive interaction” means that the interaction ofHIV-1 and the HIV-1 co-receptor would lead to the fusion of said HIV-1or HIV-1 envelope glycoprotein⁺ cell and the membrane bearing theco-receptor.

As used herein, “prevents the productive interaction” means that theamount of interaction is reduced as compared to the amount that wouldoccur without the compound. The interactions may be prevented by maskingor altering interactive regions on the co-receptor or HIV-1 or byaltering the expression, aggregation, conformation, or association stateof the co-receptor.

As used herein, “HIV-1 fusion co-receptor” means a cellular receptorthat mediates fusion between the target cell expressing the receptor andHIV-1 or an HIV-1 envelope glycoprotein⁺ cell. HIV-1 fusion co-receptorsinclude but are not limited to CCR5, CXCR4 and other chemokinereceptors.

This invention also provides a composition which inhibits fusion ofHIV-1 or an HIV-1 envelope glycoprotein⁺ cell to a target cell,comprising at least two compounds in synergistically effective amountsfor inhibiting fusion of HIV-1 or an HIV-1 envelope glycoprotein⁺ cellto a target cell, wherein at least one of the compounds prevents theproductive interaction between HIV-1 and an HIV-1 fusion co-receptor.

As used herein, “fusion” means the joining or union of the lipid bilayermembranes found on mammalian cells or viruses such as HIV-1. Thisprocess is distinguished from the attachment of HIV-1 to a target cell.Attachment is mediated by the binding of the HIV-1 exterior glycoproteinto the human CD4 receptor, which is not a fusion co-receptor.

As used herein, “inhibits” means that the amount is reduced as comparedwith the amount that would occur without the composition.

As used herein, “target cell” means a cell capable of being infected byor fusing with HIV-1 or HIV-1 infected cells.

As used herein, “chemokine” means a cytokine that can stimulateleukocyte movement. They may be characterized as either cys-cys orcys-X-cys depending on whether the two amino terminal cysteine residuesare immediately adjacent or separated by one amino acid. It includes butis not limited to RANTES, MIP-1α, MIP-10, SDF-1 or another chemokinewhich blocks HIV-1 infection.

In one embodiment of the above compositions, the co-receptor is achemokine receptor. In the preferred embodiment of the abovecompositions, the chemokine receptor is CCR5 or CXCR4. Several otherchemokine and related receptors are known to function as HIVco-receptors including but not limited to CCR2, CCR3, CCR8, STRL33,GPR-15, CX3CR1 and APJ (69).

As used herein, “chemokine receptor” means a member of a homologousfamily of seven-transmembrane spanning cell surface proteins that bindchemokines.

As used herein, “CCR5” is a chemokine receptor which binds members ofthe C-C group of chemokines and whose amino acid sequence comprises thatprovided in Genbank Accession Number 1705896 and related polymorphicvariants.

As used herein, “CXCR4” is a chemokine receptor which binds members ofthe C-X-C group of chemokines and whose amino acid sequence comprisesthat provided in Genbank Accession Number 400654 and related polymorphicvariants.

In one embodiment of the above compositions, at least one of thecompounds is a nonpeptidyl molecule. In one embodiment, the nonpeptidylmolecule is the bicyclam compound AMD3100. (16).

As used herein, “nonpeptidyl molecule” means a molecule that does notconsist in its entirety of a linear sequence of amino acids linked bypeptide bonds. A nonpeptidyl molecule may, however, contain one or morepeptide bonds.

In one embodiment of the above compositions, at least one of thecompounds is an antibody. In one embodiment, the antibody is amonoclonal antibody. In another embodiment, the antibody is aanti-chemokine receptor antibody. In one embodiment, the antibody is ananti-CXCR4 antibody. In a further embodiment, the anti CXCR4 antibody is12G5. (43). In a preferred embodiment, the antibody is an anti-CCR5antibody. The anti-CCR5 antibody includes but is not limited to PA8,PA9, PA10, PA11, PA12, PA14 and 2D7. In this composition the compoundsare in an appropriate ratio. The ratio ranges from 1:1 to 1000:1.

The monoclonal antibodies PA8, PA9, PA10, PA11, PA12 and PA14 weredeposited pursuant to and in satisfaction of, the requirements of theBudapest Treaty on the international Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure with the AmericanType Culture Collection (ATCC), 10801 University Boulevard, Manassas,Va. 20110-2209 on Dec. 2, 1998 under the following Accession Nos.: ATCCAccession No. HB-12605 (PA8), ATCC Accession No. HB-12606 (PA9), ATCCAccession No. HB-12607 (PA10), ATCC Accession No. HB-12608 (P11), ATCCAccession No. HB-12609 (PA12) ATCC Accession No. HB-12610 (PA14).

In another embodiment of the above compositions, two or more of thecompounds are antibodies. In one embodiment of the invention, theantibodies include but are not limited to PA8, PA9, PA10, PA11, PA12,PA14 and 2D7. In this composition the antibodies are in an appropriateratio. The ratio ranges from 1:1 to 50:1.

As used herein, “antibody” means an immunoglobulin molecule comprisingtwo heavy chains and two light chains and which recognizes an antigen.The immunoglobulin molecule may derive from any of the commonly knownclasses, including but not limited to IgA, secretory IgA, IgG and IgM.IgG subclasses are also well known to those in the art and include butare not limited to human IgG1, IgG2, IgG3 and IgG4. It includes, by wayof example, both naturally occurring and non-naturally occurringantibodies. Specifically, “antibody” includes polyclonal and monoclonalantibodies, and monovalent and divalent fragments thereof. Furthermore,“antibody” includes chimeric antibodies, wholly synthetic antibodies,single chain antibodies, and fragments thereof. Optionally, an antibodycan be labeled with a detectable marker. Detectable markers include, forexample, radioactive or fluorescent markers. The antibody may be a humanor nonhuman antibody. The nonhuman antibody may be humanized byrecombinant methods to reduce its immunogenicity in man. Methods forhumanizing antibodies are known to those skilled in the art.

As used herein, “monoclonal antibody,” also designated as mAb, is usedto describe antibody molecules whose primary sequences are essentiallyidentical and which exhibit the same antigenic specificity. Monoclonalantibodies may be produced by hybridoma, recombinant, transgenic orother techniques known to one skilled in the art.

As used herein, “anti-chemokine receptor antibody” means an antibodywhich recognizes and binds to an epitope on a chemokine receptor. Asused herein, “anti-CCR5 antibody” means a monoclonal antibody whichrecognizes and binds to an epitope on the CCR5 chemokine receptor.

As used herein, “appropriate ratio” means mass or molar ratios whereinthe compounds are synergistically effective.

In one embodiment of the above compositions, at least one compound is achemokine or chemokine derivative. The chemokines include but are notlimited to RANTES, MIP-1α, MIP-1β, SDF-1 or a combination thereof. Inthis composition, the compounds are in an appropriate ratio. Thechemokine derivatives include but are not limited to Met-RANTES,AOP-RANTES, RANTES 9-68, or a combination thereof.

As used herein, “chemokine derivative” means a chemically modifiedchemokine. The chemical modifications include but are not limited toamino acid substitutions, additions or deletions, non-peptidyl additionsor oxidations. One skilled in the art will be able to make suchderivatives.

In another embodiment of the above compositions, at least one compoundis an antibody and at least one compound is a chemokine or chemokinederivative. In this composition, the compounds are in an appropriateratio. The ratio ranges from 100:1 to 1000:1.

In another embodiment of the above compositions, at least one compoundbinds to the gp41 subunit of the HIV-1 envelope glycoprotein. In oneembodiment, at least one compound is the T-20 peptide inhibitor of HIV-1entry (70).

In another embodiment of the above compositions, at least one of thecompounds inhibits the attachment of HIV-1 to a target cell. In oneembodiment, at least one compound binds CD4. In one embodiment, at leastone compound is an HIV-1 envelope glycoprotein. In one embodiment, atleast one compound is an anti-CD4 antibody. In one embodiment, at leastone compound binds to the HIV-1 envelope glycoprotein. In oneembodiment, at least one compound is an antibody to the HIV-1 envelopeglycoprotein. In one embodiment, at least one compound is a CD4-basedprotein. In one embodiment, at least one compound is CD4-IgG2.

In another embodiment of the above compositions, at least one compoundis an antibody and at least one compound binds to an HIV-1 envelopeglycoprotein. In one embodiment, the compound is a CD4-based protein. Inone embodiment, the compound is CD4-IgG2. In this composition, thecompounds are in an appropriate ratio. The ratio ranges from 1:1 to10:1.

As used herein, “attachment” means the process that is mediated by thebinding of the HIV-1 envelope glycoprotein to the human CD4 receptor,which is not a fusion co-receptor.

As used herein, “CD4” means the mature, native, membrane-bound CD4protein comprising a cytoplasmic domain, a hydrophobic transmembranedomain, and an extracellular domain which binds to the HIV-1 gp120envelope glycoprotein.

As used herein, “HIV-1 envelope glycoprotein” means the HIV-1 encodedprotein which comprises the gp120 surface protein, the gp41transmembrane protein and oligomers and precursors thereof.

As used herein, “CD4-based protein” means any protein comprising atleast one sequence of amino acid residues corresponding to that portionof CD4 which is required for CD4 to form a complex with the HIV-1 gp120envelope glycoprotein.

As used herein, “CD4-IgG2” means a heterotetrameric CD4-human IgG2fusion protein encoded by the expression vectors deposited under ATCCAccession Numbers 75193 and 75194.

In one embodiment of the above compositions at least one of thecompounds comprises a polypeptide which binds to a CCR5 epitope. In oneembodiment, the epitope is located in the N-terminus, one of the threeextracellular loop regions or a combination thereof. In one embodiment,the epitope is located in the N-terminus. The epitope can comprise N13and Y15 in the N-terminus. The epitope can comprise comprises Q4 in theN-terminus. In another embodiment, the epitope includes residues in theN-terminus and second extracellular loop. The epitope can comprise D2,Y3, Q4, S7, P8 and N13 in the N-terminus and Y176 and T177 in the secondextracellular loop. The epitope can comprise D2, Y3, Q4, P8 and N13 inthe N-terminus and Y176 and T177 in the second extracellular loop. Theepitope can comprise D2 in the N-terminus and R168 and Y176 in thesecond extracellular loop. In one embodiment, the epitope is located inthe second extra cellular loop. The epitope can comprise Q170 and K171in the second extracellular loop. The epitope can comprise Q170 and E172in the second extra cellular loop.

As used herein, the following standard abbreviations are used throughoutthe specification to indicate specific amino acids:

-   -   A=ala=alanine R=arg=arginine    -   N=asn=asparagine D=asp=aspartic acid    -   C=cys=cysteine Q=gln=glutamine    -   E=glu=glutamic acid G=gly=glycine    -   H=his=histidine I=ile=isoleucine    -   L=leu=leucine K=lys=lysine    -   M=met=methionine F=phe=phenylalanine    -   P=pro=proline S=ser=serine    -   T=thr=threonine W=trp=tryptophan    -   Y=tyr=tyrosine V=val=valine

As used herein, “polypeptide” means two or more amino acids linked by apeptide bond.

As used herein, “epitope” means a portion of a molecule or moleculesthat forms a surface for binding antibodies or other compounds. Theepitope may comprise contiguous or noncontiguous amino acids,carbohydrate or other nonpeptidyl moities or oligomer-specific surfaces.

As used herein, “N-terminus” means the sequence of amino acids spanningthe initiating methionine and the first transmembrane region.

As used herein, “second extra cellular loop” means the sequence of aminoacids that span the fourth and fifth transmembrane regions and arepresented on the surface.

In one embodiment of the above compositions at least one of thecompounds comprises a light chain of an antibody. In another embodimentof the above compositions at least one of the compounds comprises aheavy chain of an antibody. In another embodiment of the abovecompositions at least one of the compounds comprises the Fab portion ofan antibody. In another embodiment of the above compositions at leastone of the compounds comprises the variable domain of an antibody. Inanother embodiment, the antibody is produced as a single polypeptide or“single chain” antibody which comprises the heavy and light chainvariable domains genetically linked via an intervening sequence of aminoacids. In another embodiment of the above compositions at least one ofthe compounds comprises one or more CDR portions of an antibody.

As used herein, “heavy chain” means the larger polypeptide of anantibody molecule composed of one variable domain (VH) and three or fourconstant domains (CH1, CH₂, CH3, and CH4), or fragments thereof.

As used herein, “light chain” means the smaller polypeptide of anantibody molecule composed of one variable domain (VL) and one constantdomain (CL), or fragments thereof.

As used herein, “Fab” means a monovalent antigen binding fragment of animmunoglobulin that consists of one light chain and part of a heavychain. It can be obtained by brief papain digestion or by recombinantmethods.

As used herein, “F(ab′)₂ fragment” means a bivalent antigen bindingfragment of an immunoglobulin that consists of both light chains andpart of both heavy chains. It can be obtained by brief pepsin digestionor recombinant methods.

As used herein, “CDR” or “complementarity determining region” means ahighly variable sequence of amino acids in the variable domain of anantibody.

This invention provides the above compositions and a pharmaceuticallyacceptable carrier. Pharmaceutically acceptable carriers are well knownto those skilled in the art. Such pharmaceutically acceptable carriersmay include but are not limited to aqueous or non-aqueous solutions,suspensions, and emulsions. Examples of non-aqueous solvents arepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,and injectable organic esters such as ethyl oleate. Aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,saline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, inert gases and the like.

This invention provides a method of treating a subject afflicted withHIV-1 which comprises administering to the subject an effective dose ofthe above compositions.

As used herein, “subject” means any animal or artificially modifiedanimal capable of becoming HIV-infected. Artificially modified animalsinclude, but are not limited to, SCID mice with human immune systems.The animals include but are not limited to mice, rats, dogs, guineapigs, ferrets, rabbits, and primates. In the preferred embodiment, thesubject is a human.

As used herein, “treating” means either slowing, stopping or reversingthe progression of an HIV-1 disorder. In the preferred embodiment,“treating” means reversing the progression to the point of eliminatingthe disorder. As used herein, “treating” also means the reduction of thenumber of viral infections, reduction of the number of infectious viralparticles, reduction of the number of virally infected cells, or theamelioration of symptoms associated with HIV-1.

As used herein, “afflicted with HIV-1” means that the subject has atleast one cell which has been infected by HIV-1.

As used herein, “administering” may be effected or performed using anyof the methods known to one skilled in the art. The methods may compriseintravenous, intramuscular or subcutaneous means.

The dose of the composition of the invention will vary depending on thesubject and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg. Based upon the composition, thedose can be delivered continuously, such as by continuous pump, or atperiodic intervals. For example, on one or more separate occasions.Desired time intervals of multiple doses of a particular composition canbe determined without undue experimentation by one skilled in the art.

As used herein, “effective dose” means an amount in sufficientquantities to either treat the subject or prevent the subject frombecoming HIV-1 infected. A person of ordinary skill in the art canperform simple titration experiments to determine what amount isrequired to treat the subject.

This invention provides a method of preventing a subject fromcontracting HIV-1 which comprises administering to the subject aneffective dose of the above compositions.

As used herein, “contracting HIV-1” means becoming infected with HIV-1,whose genetic information replicates in and/or incorporates into thehost cells.

This invention provides an anti-CCR5 monoclonal antibody. The antibodyincludes but is not limited to the following: PA8 (ATCC Accession No.HB-12605), PA9 (ATCC Accession No. HB-12606), PA10 (ATCC Accession No.HE-12607), PA11 (ATCC Accession No. HB-12608), PA12 (ATCC Accession No.HE-12609), and PA14 (ATCC Accession No. HB-12610).

This invention provides humanized forms of the above antibodies.

As used herein, “humanized” describes antibodies wherein some, most orall of the amino acids outside the CDR regions are replaced withcorresponding amino acids derived from human immunoglobulin molecules.In one embodiment of the humanized forms of the antibodies, some, mostor all of the amino acids outside the CDR regions have been replacedwith amino acids from human immunoglobulin molecules but where some,most or all amino acids within one or more CDR regions are unchanged.Small additions, deletions, insertions, substitutions or modificationsof amino acids are permissible as long as they would not abrogate theability of the antibody to bind a given antigen. Suitable humanimmunoglobulin molecules would include IgG1, IgG2, IgG3, IgG4, IgA andIgM molecules. A “humanized” antibody would retain a similar antigenicspecificity as the original antibody, i.e., in the present invention,the ability to bind CCR5.

One skilled in the art would know how to make the humanized antibodiesof the subject invention. Various publications, several of which arehereby incorporated by reference into this application, also describehow to make humanized antibodies. For example, the methods described inU.S. Pat. No. 4,816,567 (71) comprise the production of chimericantibodies having a variable region of one antibody and a constantregion of another antibody.

U.S. Pat. No. 5,225,539 (72) describes another approach for theproduction of a humanized antibody. This patent describes the use ofrecombinant DNA technology to produce a humanized antibody wherein theCDRs of a variable region of one immunoglobulin are replaced with theCDRs from an immunoglobulin with a different specificity such that thehumanized antibody would recognize the desired target but would not berecognized in a significant way by the human subject's immune system.Specifically, site directed mutagenesis is used to graft the CDRs ontothe framework.

Other approaches for humanizing an antibody are described in U.S. Pat.Nos. 5,585,089 (73) and 5,693,761 (74) and WO 90/07861 which describemethods for producing humanized immunoglobulins. These have one or moreCDRs and possible additional amino acids from a donor immunoglobulin anda framework region from an accepting human immunoglobulin. These patentsdescribe a method to increase the affinity of an antibody for thedesired antigen. Some amino acids in the framework are chosen to be thesame as the amino acids at those positions in the donor rather than inthe acceptor. Specifically, these patents describe the preparation of ahumanized antibody that binds to a receptor by combining the CDRs of amouse monoclonal antibody with human immunoglobulin framework andconstant regions. Human framework regions can be chosen to maximizehomology with the mouse sequence. A computer model can be used toidentify amino acids in the framework region which are likely tointeract with the CDRs or the specific antigen and then mouse aminoacids can be used at these positions to create the humanized antibody.

The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 (75)also propose four possible criteria which may used in designing thehumanized antibodies. The first proposal was that for an acceptor, use aframework from a particular human immunoglobulin that is unusuallyhomologous to the donor immunoglobulin to be humanized, or use aconsensus framework from many human antibodies. The second proposal wasthat if an amino acid in the framework of the human immunoglobulin isunusual and the donor amino acid at that position is typical for humansequences, then the donor amino acid rather than the acceptor may beselected. The third proposal was that in the positions immediatelyadjacent to the 3 CDRs in the humanized immunoglobulin chain, the donoramino acid rather than the acceptor amino acid may be selected. Thefourth proposal was to use the donor amino acid reside at the frameworkpositions at which the amino acid is predicted to have a side chain atomwithin 3A of the CDRs in a three dimensional model of the antibody andis predicted to be capable of interacting with the CDRs. The abovemethods are merely illustrative of some of the methods that one skilledin the art could employ to make humanized antibodies. The affinityand/or specificity of the binding of the humanized antibody may beincreased using methods of directed evolution as described in Wu et al.(1999) J. Mol. Biol. 284:151 and U.S. Pat. Nos. 6,165,793; 6,365,408 and6,413,774.

In an embodiment of the invention the humanized form of the antibodycomprises a light chain variable amino acid sequence as set forth in SEQID NO:6. In another embodiment, the antibody comprises a heavy chainvariable amino acid sequence as set forth in SEQ ID NO:9. In a furtherembodiment, the antibody may comprise the heavy chain variable aminoacid sequence as set forth in SEQ ID NO:12.

In another embodiment, the humanized antibody comprises the light chainvariable amino acid sequence as set forth in SEQ ID NO:6, and the heavychain variable amino acid sequence as set forth in SEQ ID NO:9.Alternatively, the antibody may comprise the light chain variable aminoacid sequence as set forth in SEQ ID NO:6 and the heavy chain variableamino acid sequence as set forth in SEQ ID NO:12.

The variable regions of the humanized antibody may be linked to at leasta portion of an immunoglobulin constant region of a humanimmunoglobulin. In one embodiment, the humanized antibody contains bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3 and sometimes, CH4 region.In one embodiment, the constant regions of the humanized antibody are ofthe human IgG4 isotype.

This invention provides isolated nucleic acid molecules encoding theseanti-CCR5 monoclonal antibodies or their humanized versions. The nucleicacid molecule can be RNA, DNA or cDNA. In one embodiment, the nucleicacid molecule encodes the light chain. In one embodiment, the nucleicacid molecule encodes the heavy chain. In one embodiment, the nucleicacid encodes both the heavy and light chains. In one embodiment, one ormore nucleic acid molecules encode the Fab portion. In one embodiment,one or more nucleic acid molecules encode CDR portions. In oneembodiment, the nucleic acid molecule encodes the variable domain. Inanother embodiment, the nucleic acid molecule encodes the variabledomain and one or more constant domains.

Preferably, analogs of exemplified humanized anti-CCR5 antibodies differfrom exemplified humanized anti-CCR5 antibodies by conservative aminoacid substitutions. For purposes of classifying amino acid substitutionsas conservative or non-conservative, amino acids may be grouped asfollows: Group I (hydrophobic side chains): met, ala, val, leu, ile;Group II (neutral hydrophilic side chains): cys, ser, thr; Group III(acidic side chains): asp, glu; Group IV (basic side chains): asn, gsr,his, lys, arg; Group V (residues influencing chain orientation): gly,pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Analogs of humanized anti-CCR5 antibodies show substantial amino acidsequence identity with humanized PRO 140 #1 or humanized PRO 140 #2,exemplified herein. Heavy and light chain variable regions of analogsare encoded by nucleic acid sequences that hybridize with the nucleicacids encoding the heavy or light chain variable regions of humanizedPRO 140 #1, or humanized PRO 140 #2, or degenerate forms thereof, understringent conditions.

Due to the degeneracy of the genetic code, a variety of nucleic acidsequences encode the humanized anti-CCR5 antibody of the presentinvention. In certain embodiments, the antibody is encoded by a nucleicacid molecule that is highly homologous to the foregoing nucleic acidmolecules. Preferably the homologous nucleic acid molecule comprises anucleotide sequence that is at least about 90% identical to thenucleotide sequence provided herein. More preferably, the nucleotidesequence is at least about 95% identical, at least about 97% identical,at least about 98% identical, or at least about 99% identical to thenucleotide sequence provided herein. The homology can be calculatedusing various, publicly available software tools well known to one ofordinary skill in the art. Exemplary tools include the BLAST systemavailable from the website of the National Center for BiotechnologyInformation (NCBI) at the National Institutes of Health.

One method of identifying highly homologous nucleotide sequences is vianucleic acid hybridization. Thus the invention also includes humanizedCCR5 antibodies having the CCR5-binding properties and other functionalproperties described herein, which are encoded by nucleic acid moleculesthat hybridize under high stringency conditions to the foregoing nucleicacid molecules. Identification of related sequences can also be achievedusing polymerase chain reaction (PCR) and other amplification techniquessuitable for cloning related nucleic acid sequences. Preferably, PCRprimers are selected to amplify portions of a nucleic acid sequence ofinterest, such as a CDR.

The term “high stringency conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. One example of highstringency conditions is hybridization at 65 degrees Centigrade inhybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄ (pII7), 0.5%SDS, 2 mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate,pH7; SDS is sodium dodecyl sulphate; and EDTA isethylenediaminetetracetic acid. After hybridization, a membrane uponwhich the nucleic acid is transferred is washed, for example, in 2×SSCat room temperature and then at 0.1-0.5×SSC/0.1×SDS at temperatures upto 68 degrees Centigrade.

The nucleic acid sequences are expressed in hosts after the sequenceshave been operably linked to (i.e., positioned to ensure the functioningof) an expression control sequence. These expression vectors aretypically replicable in the host organisms, either as episomes or as anintegral part of the host chromosomal DNA. Commonly, expression vectorswill contain selection markers, e.g., tetracycline or neomycin, topermit detection of those cells transformed with the desired DNAsequences (see, e.g., U.S. Pat. No. 4,704,362 which is incorporatedherein by reference).

E. coli is one prokaryotic host useful particularly for cloning the DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilus, and otherenterobacteriaccae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, may also be useful for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(see, Winnacker, “From Genes to Clones,”, VCH Publishers, New York, N.Y.(1987)). Eukaryotic cells are actually preferred, because a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed in the art, and include the CHO cell lines, variousCOS cell lines, HeLa cells, preferably myeloma cell lines, etc. andtransformed B cells or hybridomas. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen, et al., Immunol. Rev., 89,49-68 (1986) which is incorporated herein by reference), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, Adenovirus, cytomegalovirus, BovinePapilloma Virus, and the like.

The vectors containing the DNA segments of interest (e.g., the heavy andlight chain encoding sequences and expression control sequences) can betransferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment or electroporation may be used for other cellularhosts (see generally, Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press (1982) which is incorporated herein byreference).

Once expressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see generally, R. Scopes, “ProteinPurification”, Springer-Verlag, New York (1982)). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred, for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically (includingextracorporeally) or in developing and performing assay procedures,immunofluorescent stainings and the like (see generally, ImmunologicalMethods, Vols. I and II, Lefkovits and Pernis, eds., Academic Press, NewYork, N.Y. (1979 and 1981)).

For diagnostic or detection purposes, the antibodies may either belabeled or unlabeled. Unlabeled antibodies can be used in combinationwith other labeled antibodies (second antibodies) that are reactive withthe humanized antibody, such as antibodies specific for humanimmunoglobulin constant regions. Alternatively, the antibodies can bedirectly labeled. A wide variety of labels can be employed, such asradionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands (particularly haptens), etc. Numerous typesof immunoassays are available and are well known to those skilled in theart for detection of CCR5-expressing cells or detection of CCR5modulation on cells capable of expressing CCR5.

The present invention also provides antibody fragment-polymer conjugateshaving an effective size or molecular weight that confers an increase inserum half-life, an increase in mean residence time in circulation (MRT)and/or a decrease in serum clearance rate over underivatized antibodyfragments.

The antibody fragment-polymer conjugates of the invention can be made byderivatizing the desired antibody fragment with an inert polymer. Itwill be appreciated that any inert polymer which provides the conjugatewith the desired apparent size or which has the selected actualmolecular weight is suitable for use in constructing the antibodyfragment-polymer conjugates of the invention.

Many inert polymers are suitable for use in pharmaceuticals. See, e.g.,Davis et al., Biomedical Polymers: Polymeric Materials andPharmaceuticals for Biomedical Use, pp. 441-451 (1980). In allembodiments of the invention, a non-protinaceous polymer is used. Thenonprotinaceous polymer ordinarily is a hydrophilic synthetic polymer,i.e., a polymer not otherwise found in nature. However, polymers whichexist in nature and are produced by recombinant or in vitro methods arealso useful, as are polymers which are isolated from native sources.Hydrophilic polyvinyl polymers fall within the scope of this invention,e.g., polyvinylalcohol and polyvinvypyrrolidone. Particularly useful arepolyalkylene ethers such as polyethylene glycol (PEG); polyoxyalklyenessuch as polyoxyethylene, polyoxypropylene and block copolymers ofpolyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates;carbomers; branched or unbranched polysaccharides which comprise thesaccharide monomers D-mannose, D- and L-galactose, fucose, fructose,D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid (e.g., polymannuronic acid, or alginic acid),D-glucosamine, D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g., hyaluronic acid, polymers of sugar alcoholssuch as polysorbitol and polymannitol, heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble butthe final conjugate must be water soluble. Preferably, the conjugateexhibits a water solubility of at least about 0.01 mg/ml and morepreferably at least about 0.1 mg/ml, and still more preferably at leastabout 1 mg/ml. In addition the polymer should not be highly immunogenicin the conjugate form, nor should it possess viscosity that isincompatible with intravenous infusion or injection if the conjugate isintended to be administered by such routes.

In one embodiment, the polymer contains only a single group which isreactive. This helps to avoid cross-linking of protein molecules.However it is within the scope of the invention to maximize reactionconditions to reduce cross-linking, or to purify the reaction productsthrough gel filtration or ion-exchange chromatography to recoversubstantially homogeneous derivatives. In other embodiments the polymercontains two or more reactive groups for the purpose of linking multipleantibody fragments to the polymer backbone. Again, gel filtration orion-exchange chromatography can be used to recover the desiredderivative in substantially homogeneous form.

The molecular weight of the polymer can range up to about 500,000 D andpreferably is at least about 20,000 D, or at least about 30,000 D, or atleast about 40,000 D. The molecular weight chosen can depend upon theeffective size of the conjugate to be achieved, the nature (e.g.,structure such as linear or branched) of the polymer and the degree ofderivitization, i.e., the number of polymer molecules per antibodyfragment, and the polymer attachment site or sites on the antibodyfragment.

The polymer can be covalently linked to the antibody fragment through amultifunctional crosslinking agent which reacts with the polymer and oneor more amino acid residues of the antibody fragment to be linked.However, it is also within the scope of the invention to directlycrosslink the polymer by reacting a derivatized polymer with theantibody fragment, or vice versa.

The covalent crosslinking site on the antibody fragment includes theN-terminal amino group and epsilon amino groups found on lysineresidues, as well other amino, imino, carboxyl, sulfhydryl, hydroxyl orother hydrophilic groups. The polymer may be covalently bonded directlyto the antibody fragment without the use of a multifunctional(ordinarily bifunctional) crosslinking agent, as described in U.S. Pat.No. 6,458,355.

The degree of substitution with such a polymer will vary depending uponthe number of reactive sites on the antibody fragment, the molecularweight, hydrophilicity and other characteristics of the polymer, and theparticular antibody fragment derivitization sites chosen. In general,the conjugate contains from 1 to about 10 polymer molecules, but greaternumbers of polymer molecules attached to the antibody fragments of theinvention are also contemplated. The desired amount of derivitization iseasily achieved by using an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the level of polymer substitution ofthe conjugates is determined by size exclusion chromatography or othermeans known in the art.

Functionalized PEG polymers to modify the antibody fragments of theinvention are available from Shearwater Polymers, Inc. (Huntsville,Ala.). Such commercially available PEG derivatives include, but are notlimited to, amino-PEG, PEG amino acid esters, PEG-hydrazide, PEG-thiol,PEG-succinate, carboxymethylated PEG, PEG-propionic acid, PEG aminoacids, PEG succinimidyl succinate, PEG succinimidyl propionate,succinimidyl ester of carboxymethylated PEG, succinimidyl carbonate ofPEG, succinimidyl esters of amino acid PEGs, PEG-oxycarbonylimidazole,PEG-nitrophenyl carbonate, PEG tresylate, PEG-glycidyl ether,PEG-aldehyde, PEG-vinylsulfone, PEG-maleimide,PEG-orthopyridyl-disulfide, heterofunctional PEGS, PEG vinylderivatives, PEG silanes and PEG phospholides. The reaction conditionsfor coupling these PEG derivatives will vary depending on the protein,the desired degree of PEGylation and the PEG derivative utilized. Somefactors involved in the choice of PEG derivatives include: the desiredpoint of attachment (such as lysine or cysteine R-groups), hydrolyticstability and reactivity of the derivatives, stability, toxicity andantigenicity of the linkage, suitability for analysis, etc. Specificinstructions for the use of any particular derivative are available fromthe manufacturer. The conjugates of this invention are separated fromthe unreacted starting materials by gel filtration or ion exchange HPLC.

The anti-CCR5 antibody or fragments thereof may be used in combinationwith one or more additional anti-viral agents selected from the groupconsisting of nonnucleoside reverse transcriptase inhibitors (NNRTIs), anucleoside reverse transcriptase inhibitor, an HIV-1 protease inhibitor,a viral entry inhibitor and combinations thereof.

The known NNRTI compounds that may be used in the composition of thepresent invention include but are not limited to efavirenz, UC-781, HBY097, nevirapine(11-cyclopropyl-5,11,-dihydro-4-methyl-6H-dipyrido[3,2-b:2′3′-][1,4]diazepin-6-one),delavirdine ((Rescriptor™; Pharmacia Upjohn) (piperazine,1-[3-[(1-methyl-ethyl)amino]-2-pyridinyl]-4-[[5-[(methylsulfonyl)amino]-1H-indol-2-yl]carbonyl]-,monomethanesulfonate), SJ-3366(1-(3-cyclopenten-1-yl)methyl-6-(3,5-dimethylbenzoyl)-5-ethyl-2,4-pyrimidinedione),MKC-442 (6-benzyl-1-(ethoxymethyl)-5-isopropyluracil), GW420867x (S-3ethyl-6-fluoro-4-isopropoxycarbonyl-3,4-dihydro-quinoxalin-2(1H)-one;Glaxo), HI-443(N′-[2-(2-thiophene)ethyl]-N′-[2-(5-bromopyridyl)]-thiourea), and thelike.

The nucleoside reverse transcriptase inhibitors that may be used in thecomposition in combination with at least one anti-CCR5 antibody orfragment thereof of the present invention include but are not limited toabacavir (Ziagen™, GlaxoSmithKline)((1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolsulfate (salt)), lamivudine (Epivir™, GlaxoSmthKline)((2R,cis)-4-amino-1-(2-hydroxymethyl-1,3-oxathiolan-5-yl)-(1H)-pyrimidin-2-one),zidovudine (Retrovir™; GlaxoSmithKline) (3′azido-3′-deoxythymidine),stavudine (Zerit; Bristol-Myers Squibb)(2′,3′-didehydro-3′deoxythymidine), zacitabine (Hivid™; RocheLaboratories)(4-amino-1-beta-D2′,3′-dideoxyribofuranosyl-2-(1H)-pyrimidone),didanosine, and the like.

The HIV-1 protease inhibitors that may be used in the composition incombination with anti-CCR5 antibody or fragments thereof of the presentinvention include but are not limited to lopinavir (1S-[1R*,(R*),3R*,4R*]]-N-4-[[(2,6-dimethylphenoxy)acetyl]amino]-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]tetrahydro-alpha-(1-methylethyl)-2-oxol(2H)-pyrimidineacetamide),saquinavir(N-tert-butyl-decahydro-2-[2(R)-hydroxy-4-phenyl-3(S)-[[N-(2-quinolylcarbonyl)-L-asparaginyl]amino]butyl]-(4aS,8aS)-isoquinoline-(3S)-carboxamide),nelfinavir mesylate([3S-[2(2S*,3S*),3a,4β,8aβ]]-N-(1,1-dimethylethyl)decahydro-2[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamidemono-methane sulfonate), indinavir sulfate(([1(1S,2R),5(S))]-2,3,5-trideoxy-N-(2,3-dihydro-2-hydroxy-1H-inden-1-yl)-5-[2-[[(1,1-dimethylethyl)amino]carbonyl]-4-(3-pyridinylmethyl)-1-piperazinyl]-2-(phenylmethyl)-D-erythropentonamidesulfate (1:1) salt), amprenavir ((3S)-tetrahydro-3-furylN-[(1S,2R)-3-(4-amino-N-isobutylbenzenesulfonamido)-1-benzyl-2-hydroxypropyl]carbamate),ritonavir((10-Hydroxy-2-methyl-5-(1-methylethyl)-1-[2-(1-methylethyl)-4-thiazolyl]-3,6-dioxo-8,11-bis(phenylmethyl)-2,4,7,12-tetraazamidecan-13-oicacid,5-thiazolylmethyl ester, [5S-(5R*,8R*,10R*,11R*)]), and the like.

HIV-1 fusion or viral entry inhibitors that may be used in combinationwith the anti-CCR5 antibody or fragments thereof of the presentinvention include PRO 542 (Progenics Pharmaceuticals, Inc., Tarrytown,N.Y.), T-20 (Trimeris, Inc., Durham, N.C.) (U.S. Pat. Nos. 5,464,933;6,133,418; 6,020,459), T-1249 (U.S. Pat. Nos. 6,345,568; 6,258,782), andthe like.

For combination therapy, the anti-CCR5 antibody or fragment thereof ofthe present invention may be provided to the subject prior to,subsequent to, or concurrently with one or more conventional antiviralagents.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Example 1 A. Materials and Methods 1) Reagents

MAb 2D7 was purchased from Pharmingen (San Diego, Calif.) and CC- andCXC-chemokines were obtained from R&D Systems (Minneapolis, Minn.).CD4-IgG2 (1), soluble (s) CD4 (2) and recombinant HIV-1_(JR-FL) gp120,were produced by Progenics Pharmaceuticals, Inc. (59).

2) Isolation and Purification of Anti-CCR5 mAbs

L1.2-CCR5 cells (63) were incubated for 16 h in the presence of 5 mMsodium butyrate, which activates transcription from the cytomegalovirus(CMV) promoter that controls CCR5 expression, resulting in a 10-foldincrease in cell surface co-receptor density. Female Balb/c mice wereimmunized intraperitoneally with 10⁷ L1.2-CCR5⁺ cells at 3-weekintervals, and administered an intravenous boost of 10⁷ L1.2-CCR5⁺ cellsthree days prior to splenectomy. Splenocytes were fused with the Sp2/0cell line. In a primary screen, supernatants from ten thousand hybridomacultures were tested; one hundred and twenty of these inhibited HIV-1envelope-mediated fusion between PM1 cells (10), which naturally expressCCR5 and CD4, and HeLa-Env_(JR-FL) ⁺ cells in a resonance energytransfer (RET) assay, as previously described (19, 38). Hybridomas thatproduced the most potently inhibitory supernatants and that also stainedCCR5⁺ cells were sub-cloned by limiting dilution. Ascites fluids wereprepared by Harlan Bioproducts for Science, Inc. (Indianapolis, Ind.)from Balb/c mice that were injected with hybridomas producing theanti-CCR5 mAbs PA8, PA9, PA10, PA11, PA12 and PA14. The mAbs wereindividually purified to >95% homogeneity by precipitation with ammoniumsulfate followed by protein-A chromatography. All mAbs were resuspendedin phosphate buffered saline (PBS) at a final concentration of 5 mg/ml.

3) Fluorescence Activated Cell Sorting (FACS) Analysis and EpitopeMapping of Anti-CCR5 mAbs

Flow cytometry was used to detect cell-surface reactivity of mAbsPA8-PA12 and PA14 with CCR5. Sodium butyrate treated L1.2-CCR5⁺ cells(10⁶) were incubated with 0.25 μg of antibody, for 20 min at 4° C. in0.1% sodium azide (NaN₂) in 50 μl of Dulbecco's PBS (DPBS). The CCR5 mAb2D7 was used as a positive control, a non-specific murine IgG1 was usedas a negative control. The cells were spun down, washed and incubatedwith phycoerythrin (PE)-labeled goat anti-mouse IgG (Caltag, Burlingame,Calif.) diluted 1:100, under the same conditions as the first antibodyincubation. Finally, cells were analyzed by flow cytometry. PBMC wereisolated and stimulated as previously described (60) and stained usingsimilar methods.

A similar procedure was used for epitope mapping of the anti-CCR5 mAbs.A panel of seventy CCR5 point mutants has been described (20, 24, 52).The coding sequences of these proteins are sub-cloned into the pcDNA3.1vector (Stratagene) from which transcription can be driven by a 5′T7-polymerase promoter. The CCR5 mutants carry a 9-residue hemaglutinin(HA) tag at the C-terminus for detection of protein in cell lysates orby flow cytometry. HeLa cells (2×10⁶) were incubated for 5 h with 20μg/ml lipofectin and an equal amount of wild-type or mutantCCR5-expressing plasmid in OPTI-MEM (Life Technologies, Gaithersburg,Md.). The cells were then infected for 12 h with 2×10⁷ p.f.u. of vTF7(23) to boost CCR5 expression, detached with 2 mM ethylenediaminetetraacetic acid (EDTA) in PBS and washed once with binding buffer (1%BSA, 0.05% NaN₃ in DPBS). Cells (1×10⁶) were surface labeled with mAbsas described in the previous paragraph, washed once with the incubationbuffer and resuspended in 1 ml of 1×FACSlyse in water (Becton Dickinson)for 30 min at room temperature, to permeabilize the cell membranes. Thecells were then spun down, washed with the incubation buffer andincubated for 1 h at 37° C. with 4 μg/ml of a fluorescein isothiocyanate(FITC)-labeled mouse anti-HA mAb (BabCo, Richmond, Calif.) forintracellular labeling. Finally, cells were washed once with bindingbuffer and once with DPBS, resuspended in 1% formaldehyde in PBS andanalyzed by flow cytometry. The extent of binding of a mAb to mutantCCR5 was determined by the equation (mutant CCR5 PE m.f.i./wt CCR5 PEm.f.i.)/(mutant CCR5 FITC m.f.i./wt CCR5 FITC m.f.i.)×100%. Thisnormalizes mAb binding for mutant co-receptor expression levels.

4) gp120/sCD4-Binding Assay

gp120 was biotinylated using NHS-biotin (Pierce, Rockford, Ill.)according to the manufacturer's instructions, and uncoupled biotin wasremoved by diafiltration. Sodium butyrate-treated L1.2-CCR5⁺ cells wereincubated with varying dilutions of an equimolar mixture of sCD4 andbiotinylated gp120, or 1.25 μg/ml of sCD4 and 2.5 μg/ml of biotinylatedgp120 in the presence of varying concentrations of anti-CCR5 mAbsPA8-PA12, PA14, 2D7 or a non-specific murine IgG1, for 1 h at roomtemperature in 0.1% NaN₂ in DPBS. Cells were washed with the incubationbuffer and incubated with streptavidin-PE (Becton Dickinson) diluted1:50, for 1 h at room temperature. Finally, cells were washed withbinding buffer and analyzed using a fluorescence plate reader(Perspective Biosystems, Framingham, Mass.).

5) Inhibition of Envelope-Mediated Cell-Cell Fusion and HIV-1 Entry byAnti-CCR5 mAbs

HIV-1 envelope-mediated fusion between HeLa-Env_(JR-FL) ⁺ and PM1 cellswas detected using the RET assay. Equal numbers (2×10⁴) of fluoresceinoctadecyl ester (F18)-labeled envelope-expressing cells and octadecylrhodamine (R18)-labeled PM1 cells were plated in 96-well plates in 15%fetal calf serum in DPBS and incubated for 4 h at 37° C. in the presenceof varying concentrations of the anti-CCR5 mAbs, PA8-PA12, PA14, 2D7 ora non-specific murine IgG1. Fluorescence RET was measured with aCytofluor plate-reader (PerSeptive Biosystems) and % RET was determinedas previously described (38).

NLluc⁺env⁺ viruses complemented in trans by envelope glycoproteins fromJR-FL or Gun-1 were produced as previously described (20).U87MG-CD4⁺CCR5⁺ cells (14) were infected with chimeric, reporter virusescontaining 50-100 ng/ml p24 in the presence of varying concentrations ofthe individual mAbs. After 2 h at 37° C., virus-containing media werereplaced by fresh, mAb-containing media. Fresh media, withoutantibodies, were added again after 12 hours. After a total of 72 h, 100μl of lysis buffer (Promega) were added to the cells and luciferaseactivity (r.l.u.) was measured as described (20). The % inhibition ofHIV-1 infection is defined as [1-(r.l.u in the presence ofantibody/r.l.u in the absence of antibody)]×100%.

6) Calcium Signaling Assays

The fluorochrome Indo-1AM (Molecular Probes, Eugene, Oreg.) was added tosodium butyrate treated L1.2-CCR5⁺ cells at a final concentration of 5μM. After incubation at 37° C. for 30 min, the cells were washed onceand resuspended in Hank's buffered saline. Cells (10⁶) were stimulatedsequentially with an anti-CCR5 mAb or PBS, followed 60 s later withRANTES. MAbs PA8-PA12 and PA14 were used at a concentration of 100μg/ml, 2D7 at 20 μg/ml and RANTES at 250 ng/ml. Calcium flux inhibitionby PA14 and 2D7 was also tested for a wide range of mAb concentrations,ranging from 0-100 μg/ml. Intracellular calcium levels were monitoredusing a Perkin-Elmer LS-50S fluorescence spectrophotometer by measuringthe ratio of fluorescence emissions at 402 nm (bound dye) and 486 nm(free dye) following excitation at 358 nm.

B. Results and Discussion 1) Isolating anti-CCR5 Monoclonal AntibodiesPA8, PA9, PA10, PA11, PA12 and PA14

It was found that peptides corresponding to the extracellular domains ofCCR5 are inefficient at raising specific, high-titer antibody responsesagainst the native, cell surface receptor (50). Balb/C mice wereimmunized, therefore, with L1.2-CCR5⁺ cells and hybridoma culturesupernatants were tested for their ability to inhibit JR-FLenvelope-mediated membrane fusion with CD4⁺CCR5⁺ PM1 cells in the RETassay (19, 38). Even though well over a hundred supernatants inhibitedcell-cell fusion by >50%, only six—designated PA8, PA9, PA10, PA11, PA12and PA14—specifically and intensely stained L1.2-CCR5⁺ but not theparental L1.2 cells, as demonstrated by flow cytometry (data not shown).Based on previous experience, it was assumed that the other mAbs capableof inhibiting cell-cell fusion were probably directed against cellsurface adhesion molecules such as LFA-1 (37). Hybridomas PA8-PA12 andPA14 were determined by isotyping ELISA (Cappell, Durham, N.C.) tosecrete IgG1 mAbs. Ascites fluids were prepared from Balb/C mice thatwere injected with the six hybridomas and the IgG1 fractions werepurified. PA8, PA9, PA11, PA12 and PA14 exhibited distinct isoelectricfocussing profiles, whereas PA10 had a very similar profile to that ofPA9 and therefore may be a second isolate of the same mAb (data notshown).

2) MAb Binding to CCR5+ Cells

None of the purified anti-CCR5 mAbs stained the parental L1.2 cell line(data not shown). However, mAbs PA9-PA12 and PA14 stained >90%, and PA8stained ˜70%, of L1.2-CCR5⁺ cells as determined by flow cytometry,showing they recognized CCR5 (FIG. 1). The anti-CCR5 mAb 2D7, which wasa positive control in our experiments, also stained >90% of L1.2-CCR5⁺cells. PA8-PA12 and PA14 are all IgG1, and react equally well with agoat anti-mouse IgG, whereas 2D7 is an IgG2a and may react differentlywith the reporter antibody. Only mean fluorescence intensities (m.f.i.)measured with mAbs PA8-PA12 and PA14 therefore are directly comparable.The rank order of mean fluorescence intensities (m.f.i.) wasPA12˜PA11>(2D7=) PA14˜PA10˜PA9>PA8. The difference between PA12 m.f.i.and PA8 m.f.i. was three-fold. Differences in staining intensity betweenPA8 and the other mAbs remained constant over a wide range ofconcentrations (data not shown) and probably do not correspond todifferences in mAb affinities for CCR5. This implies that PA8 interactsonly with a subset of CCR5 molecules present on the surface of L1.2-CCR5cells.

Compared with L1.2-CCR5+ cells, mitogen-stimulated PBMC exhibiteddifferent patterns of staining by the anti-CCR5 mAbs. 2D7 and PA14stained >20%, PA11 and PA12 stained ˜10%, PA8, PA9 and PA10 stained <5%of PBMC (FIG. 1). The mean fluorescence intensities of the stained PBMCwere about ten-fold lower than those obtained with L1.2-CCR5⁺ cells foreach mAb; their rank order was (2D7>) PA14>PA12˜PA11˜PA10˜PA9˜PA8.Again, this differed somewhat from the order of reactivities observed onCCR5 transfectants. The difference between PA9 m.f.i. and PA14 m.f.i.was seven-fold. Other groups have observed similar differences in theability of anti-CCR5 mAbs to stain stable, CCR5⁺ cell lines versus PBMC(28). This may be due to cell-specific differences in CCR5 conformation,post-translational modification or oligomerization. Alternatively,association with other cell surface molecules may differ between cells.Since an obvious choice for such a molecule would be the CD4 cellsurface antigen, which is absent from L1.2-CCR5⁺ cells and present onPBMCs, we also tested the ability PA8-PA12, PA14 and 2D7 to stain HeLacells transiently expressing CCR5 alone or with CD4. No differences wereobserved in the ability of any of the mAbs to stain cell surface CCR5 inthe presence of CD4 (data not shown). If there is an association betweenthese two proteins, it does not involve epitopes recognized by theanti-CCR5 mAbs available to us. Alternatively, an association betweenCCR5 and CD4 might only occur on primary lymphocytes.

3) Epitope Mapping of the mAbs Using CCR5 Alanine Mutants

None of the antibodies were able to detect reduced and denatured CCR5protein by Western blotting indicating that they recognizeconformationally sensitive epitopes (data not shown). MAb epitopemapping studies were performed using a panel of seventy alanine pointmutants of residues in the Nt and ECLs of CCR5. HeLa cells werelipofected with mutant or wild type CCR5 coding sequences appended withC-terminal HA tags, and infected with vTF7 (23) to boost co-receptorexpression. The cells were then incubated with the anti-CCR5 mAbs andtheir binding was revealed by a PE-labeled goat anti-mouse IgG. Asecond, intracellular stain was performed with a FITC-labeled anti-HAmAb (BabCo). This internal control allowed us to directly normalizestaining by the anti-CCR5 mAbs for mutant co-receptor expression levelson the cell surface. Hence, mAb binding to each mutant is expressed as apercentage of binding to wild-type CCR5 (FIG. 4).

Certain point mutations reduced the binding of all of the antibodies toCCR5 by >50. In general, PA8-PA12 were the most affected, PA14 and 2D7the least affected by this class of mutants, which included the cysteinepair C101A and C178A, the Nt mutants Y10A, D11A, K25A, the ECL1 mutantD95A, the ECL2 mutants K171A/E172A, Q188A, K191A/N192A, and the ECL3mutants F263A and F264A (FIG. 1). One interpretation is that theseresidues are not part of the mAb epitopes per se, but that changing themto alanines causes conformational perturbations that have a commoneffect on binding of all mAbs. We assumed that if a mutation loweredbinding of an individual mAb by >75%, and did not also lower binding ofmost of the other antibodies, the residue was probably a directcontributor to the epitope recognized by the mAb. Using these stringentguidelines, it was concluded that the seven anti-CCR5 mAbs recognizeoverlapping but distinct epitopes (FIG. 4). MAb PA8 binding to CCR5depended on N13 and Y15 in the Nt. MAb PA9 and PA10 required D2, Y3, Q4,P8 and N13 in the Nt, and Y176 and T177 in ECL2. MAb PA9 also requiredS7 in the Nt. MAb PA11 and PA12 binding depended on Q4 in the Nt. PA14required D2 in the Nt, and R168 and Y176 in ECL2. Finally, mAb 2D7required Q170 and K171/E172 in ECL2 in order to bind to CCR5.

4) Chemokine Signaling in the Presence of Anti-CCR5 mAbs

Chemokine receptor-binding agents can be antagonists or, more rarely,agonists of receptor-mediated intracellular signaling. Alternatively,they could have no effect on signaling. CCR5 is able to bind threeCC-chemokines, RANTES, MIP-1α and MIP-1β, and transduce a signal thatmodulates cytosolic calcium levels. We therefore tested theagonist/antagonist activity of various concentrations of mAbs PA8-PA12,PA14 and 2D7. Changes in intracellular calcium concentrations, (Ca²⁺)i,were measured in Indo-1-loaded L1.2-CCR5⁺ cells. None of the mAbsstimulated a change in (Ca²⁺)i, indicating that they are not agonistsfor CCR5. PA8-PA12 were also unable to inhibit Ca²⁺ fluxes induced byRANTES (FIG. 5A and data not shown), even at concentrations as high as100 μg/ml, showing they are not antagonists either. These concentrationsprovide saturating binding of the mAbs to L1.2-CCR5⁺ cells, as shown byflow cytometry and the gp120/CCR5 binding assay (FIG. 6D and data notshown). MAbs PA14 and 2D7, however, blocked calcium mobilization inducedby RANTES, although with different potencies (FIG. 5A, 5B). The IC₅₀ forPA14 calcium influx inhibition was 50 g/ml, which was approximately8-fold higher than the IC₅₀ for 2D7 (FIG. 5B). RANTES-, MIP-1α- andMIP-1β-induced calcium fluxes were each inhibited by similarconcentrations of PA14 (data not shown). None of the mAbs affectedSDF-1-induced calcium mobilization in L1.2-CCR5⁺ cells, whichendogenously express CXCR4 (data not shown). Finally, neither mAbs norCC-chemokines affected cytosolic calcium levels in parental L1.2 cells(data not shown).

5) Inhibition of CCR5 Co-Receptor Function by the mAbs

MAbs PA8-PA12 and PA14 were initially selected on the basis of theirability to inhibit HIV-1 envelope-mediated cell-cell fusion. Thisactivity was confirmed and quantified for the purified mAbs. Asexpected, all six mAbs, as well as mAb 2D7, blocked fusion betweenCD4⁺CCR5⁺ PM1 cells and HeLa-Env_(JR-FL) ⁺ cells in the RET assay. Therank order of potency was 2D7˜PA14>PA12>PA11>PA10˜PA9˜PA8 (FIG. 6A).IC₅₀ values for PA14 and 2D7 were 1.7 μg/ml and 1.6 μg/ml respectively,for PA11 and PA12 these were 25.5 μg/ml and 10.0 μg/ml respectively(FIG. 3). PA8, PA9 and PA10 inhibited fusion by only 10-15% at 300μg/ml. None of the mAbs affected fusion between PM1 cells andHeLa-Env_(LAI) ⁺ cells, which express the full length envelope proteinfrom an X4 virus (data not shown).

The ability of the different anti-CCR5 mAbs to inhibit entry of aprototypic R5 virus, JR-FL, and a R5X4 virus, Gun-1, in a single-roundof replication, luciferase-based entry assay was also tested. The rankorder of potency in the entry assay was similar to the one determined inthe cell-cell fusion assay (FIG. 6B). A >50% inhibition of JR-FL orGun-1 entry with PA8-PA11 was unable to be obtained. The IC₅₀ value forPA12 was 2.5 μg/ml. However, inhibition of entry by >60% with this mAbwas unable to be obtained. The IC₅₀ values for PA14 and 2D7 inhibitionof JR-FL entry were determined to be 0.024 and 0.026 μg/ml respectively(FIG. 3), and were 60-fold lower then those obtained in the fusionassay. Entry of dual-tropic Gun-1 was 2-3-fold more sensitive toinhibition by anti-CCR5 mAbs than JR-FL entry (data not shown).

Anti-co-receptor mAbs might inhibit envelope-mediated fusion either bydirectly affecting the gp120/CCR5 interaction or by impedingpost-binding steps involved in the formation of an active fusioncomplex. To determine the mechanism of inhibition of viral fusion andentry by PA8-PA12 and PA14, the ability of the different mAbs to blockthe gp120/CCR5 interaction was tested. For this an assay that detectsbinding to L1.2-CCR5⁺ cells of biotinylated HIV-1_(JR-FL) gp120complexed with sCD4 was used. No binding of biotinylated gp120 wasobserved in the absence of sCD4 or CCR5, or when HIV-1_(LAI) gp120 wasused (FIG. 6C).

With the exception of PA8, all mAbs abrogated gp120/sCD4 binding toL1.2-CCR5⁺ (FIG. 6D). Inhibition by PA8 saturated at ˜40%, which concurswith flow cytometry data (FIG. 1) in suggesting that this mAb binds onlyto a subset of CCR5 molecules on L1.2-CCR5⁺ cells. MAbs PA9, PA10, PA11and PA12 inhibited binding with IC₅₀ values of 0.24, 0.13, 0.33, 0.24μg/ml respectively (FIG. 3). Surprisingly, mAbs PA14 and 2D7 were thetwo least efficient inhibitors of gp120/sCD4 binding, with IC₅₀ valuesof 1.58 and 1.38 μg/ml respectively (FIG. 3). Therefore, there was nocorrelation between the ability of a mAb to inhibitgp120/CD4/CCR5-mediated membrane fusion and entry and its ability toblock gp120/sCD4 binding to the co-receptor.

6) Synergistic Inhibition of HIV-1 Fusion by Combinations of Anti-CCR5mAbs and Other Viral Entry Inhibitors

Co-receptor-specific agents may act at multiple stages of the entryprocess and exhibit non-additive effects when used in combination. Froma clinical perspective, it is important to determine the interactions ofco-receptor-specific drug candidates with endogenous chemokines, whichmay afford some level of protection against disease progression. CCR5mAbs were therefore tested in combination with each other or withRANTES, or with CD4-IgG2, which binds to HIV-1 gp120 to inhibitattachment to target cells. Dose-response curves were obtained for theagents used individually and in combination in viral fusion and entryassays. Data were analyzed using the median effect principle (9). Theconcentrations of single-agents or their mixtures required to produce agiven effect were quantitatively compared in a term known as theCombination Index (CI). A CI value greater than 1 indicates antagonism,CI˜1 indicates an additive effect, and CI<1 indicates a synergisticeffect wherein the presence of one agent enhances the effect of another.

Combinations of PA12 and 2D7 were the most potently synergistic, with CIvalues ranging between 0.02 and 0.29, depending on the ratio of theantibodies (FIG. 7 and FIG. 2). The degree of synergy is known to varywith the stoichiometry of the agents. The viral entry and fusion assayswere generally consistent in identifying mAb combinations that arehighly synergistic, PA12 and 2D7 moderately synergistic, PA12 and PA14;additive, PA11 and PA12; and weakly antagonistic, PA14 and 2D7. The lackof synergy between PA14 and 2D7 is not surprising given that these mAbscross-compete for binding to CCR5⁺ cells as determined by flow cytometry(data not shown). The observation of an additive effect of PA11 and PA12may be an indication that these mAbs bind to slightly different epitopesin CCR5, while sharing a dependency on residue Q4 in the Nt.

The ability of mAbs PA12, PA14 and 2D7 to synergize with RANTES inblocking cell-cell fusion was also tested. PA12 and RANTES combinationsexhibited moderate synergy (FIG. 2). PA14 and 2D7 exhibited no synergywith RANTES, which is consistent with these mAbs being inhibitory ofRANTES binding and signaling (FIG. 5A, 5B). Finally, we tested synergybetween mAbs PA12, PA14, 2D7 and CD4-IgG2, which interacts with gp120.We observed moderate synergy between PA12 and CD4-IgG2 but no synergybetween PA14 or 2D7 and CD4-IgG2 (FIG. 2).

EXPERIMENTAL DISCUSSION

Six murine anti-CCR5 IgG1 mAbs were isolated and characterized. WhereasPA8, PA9, PA11, PA12 and PA14 are distinct molecular species, PA9 andPA10 are indistinguishable by the analyses and therefore are probablythe same mAb. All of the mAbs that were isolated recognize complexconformational epitopes, as is often the case with mAbs raised againstnative, cell surface proteins. Epitope mapping was performed for allmAbs using a panel of CCR5 alanine point mutants. Residues that affectedbinding of all mAbs similarly were assumed to cause conformationalperturbations in the co-receptor and not to constitute part of the mAbepitopes. Only two such residues, Y10 and D11, have been shown to affectHIV-1 entry (20, 52). The PA8, PA11 and PA12 epitopes are locatedexclusively in the Nt domain. Consistent with this result, PA8 was ableto bind a biotinylated Nt peptide, containing residues D2 through R31,in an ELISA (data not shown). However, PA11 and PA12, whose bindingstrongly depended only on Q4, did not bind the Nt peptide in solution(data not shown). One possibility is that the Nt peptide does not assumethe proper conformation for recognition by PA11 and PA12, whereas PA8binding may be less conformation-dependent. Alternatively, PA11 and PA12might interact with residues that we have not mutated, or form weakbonds with amino acids located in other domains of CCR5, or bind peptidebackbone atoms whose presentation may be unchanged by mutagenesis.Antibodies PA9, PA10 and PA14 recognized epitopes that included residuesin both the Nt and ECL2 domains of CCR5, whereas the 2D7 epitope waslocated exclusively in ECL2.

The PA14 epitope comprises both D2 in the Nt and R168 in ECL2 indicatingthat these two residues are proximal to one another within the contextof a mAb footprint. They may even directly interact with one anotherthrough their opposite charges.

MAbs PA8-PA12 and PA14 stained CCR5⁺ cells with different intensitiesand in a cell type-dependent manner. All mAbs except PA8 stained >90%L1.2-CCR5⁺ cells, the highest mean fluorescence intensity being observedwith PA11 and PA12. However, PA14 and 2D7 stained the highest percentageof PBMC and also yielded the highest mean fluorescence intensities onthese cells. Hill et al. (28) have recently characterized a panel ofanti-CCR5 mAbs that similarly stained transfected cells, but only two ofeight stained PBMC, and none stained primary monocytes. A low affinityfor CCR5 probably accounted for the non-reactivity of two of the mAbswith primary cells, but this was unlikely to be the explanation for thefailure of the other four to react. In our mAb panel, we observe themost intense staining of PBMC by mAbs 2D7 and PA14 that have epitopeslocated entirely or partially in the first ten residues of ECL2. Hill etal. report, however, that mAbs specific for the Nt and ECL1 stain PBMCs,while mAbs to ECL2 and ECL3 do not stain PBMC, so a consistent patternof reactivity has not been identified. One explanation for celltype-specific staining by mAbs would be that activated PBMCs (andmonocytes) secrete CC-chemokines that bind to cell surface CCR5, maskingsome mAb epitopes. However, one would expect this to be especially truefor PA14 and 2D7, which are antagonists of chemokine-induced calciummobilization and presumably compete with CC-chemokines for binding toCCR5. Yet these mAbs stain PBMC the most intensely. Alternatively,differential CCR5 epitope exposure may reflect cell type-specificreceptor oligomerization, association with other cell-surface molecules,or different post-translational modifications such as glycosylation. Wehave shown that differences in mAb binding probably do not reflect celltype-specific differences in CD4/CCR5 interactions.

MAbs PA8-PA12 did not inhibit CC-chemokine induced calcium mobilizationin CCR5⁺ cells, nor did they mediate signaling through CCR5. MAbs 2D7and PA14 were inhibitors of CC-chemokine induced calcium mobilization,but 2D7 was almost an order of magnitude more potent than PA14. This maybe because the PA14 epitope overlaps less with the CC-chemokine bindingdomain on CCR5 than the 2D7 epitope. All of the mAbs also blocked HIV-1entry and envelope-mediated membrane fusion, but inhibition of cell-cellfusion required in some cases almost two orders of magnitude moreantibody than what was needed to block viral entry. Presumably, moregp120/CD4/CCR5 interactions as well as interactions between adhesionmolecules are established and act cooperatively during cell-cell fusion,compared to virus-cell fusion, making it more difficult to inhibit. Thisis commonly observed with antibodies to LFA-1 or to the HIV-1 envelopeglycoprotein (45, 51). PA8, PA9 and PA10 were unable to block cell-cellfusion by >15% and viral entry by >40%, even at the highest antibodyconcentrations. However, >90% inhibition of fusion could be attainedwith PA11, PA12 and PA14, and >90% inhibition of entry could be attainedwith PA14. The most potent of the six mAbs in blocking fusion and entrywas PA14, which was as effective as 2D7. Surprisingly, PA14 and 2D7 wereamong the least potent inhibitors of gp120/sCD4 binding to L1.2-CCR5⁺cells, whereas PA9-PA12 blocked with similar potencies, and PA8 wasunable to block >40% of gp120/sCD4 binding. These observations raisequestions about the nature of the CCR5 molecules presented on differentcells and about the mechanisms of inhibition of viral fusion and entry.It may be that CCR5 on L1.2 cells, used in the mAb and gp120-bindingassays, is not in an identical conformation to CCR5 on PBMC, used in themAb-binding assay, or to CCR5 on PM1 and U87MG cells used in the fusionand entry assays.

The low staining of PBMC and the partial inhibition of fusion and entryby some of our mAbs indicate that they are only able to bind to a subsetof CCR5 molecules expressed on primary lymphocytes, PM1 andU87MG-CD4⁺CCR5⁺ cell lines. Yet, other than PA8, all mAbs are able tostain >90% L1.2-CCR5⁺ cells and to completely block binding of thegp120/sCD4 complex to these cells. At least one difference betweenL1.2-CCR5⁺ and the other cells that we have used is the density ofco-receptor protein on the cell surface. Indeed, we estimate that theL1.2-CCR5⁺ cells express 10- to 100-fold more cell surface co-receptorthan PM1 and U87MG-CD4⁺CCR5⁺ cells. But when HeLa cells are engineeredto transiently express as much co-receptor as the L1.2-CCR5⁺ cell line,we are still unable to detect gp120/sCD4 binding to them (data notshown). Over-expression of CCR5 on L1.2, along with other cell-specificfactors therefore, might favor a co-receptor conformation thatprominently exposes the Nt, making it more accessible to both mAbs andgp120. Such a conformation might be induced by receptor oligomerization,by diminished or altered associations with cell surface proteins or byreceptor interactions with G proteins (25, 62). Do multipleconformations of CCR5 co-exist on the cell surface, and are they allpermissive for viral entry? The patterns of mAb reactivity would suggestso, since HIV-1 entry and fusion can occur, albeit at reduced levels, inthe presence of mAb concentrations that saturate epitopes required forgp120 binding to L1.2-CCR5⁺ cells. We favor the hypothesis that theco-receptor molecules present on L1.2-CCR5⁺ cells possess one HIV-1entry-competent conformation whereas CCR5 molecules on PBMC, PM1 andCCR5⁺ U87MG exist in multiple, entry-competent states that displaydifferent mAb reactivities. Whereas PA14 and 2D7 may recognize allconformations, other mAbs may not. Why L1.2 cells are conducive to aparticular fusion-competent conformation remains to be determined.

It has recently been demonstrated that the gp120-binding domain lies inthe first twenty residues of the CCR5 Nt domain. MAbs to thegp120-binding domain on CCR5 potently block this interaction but are notnearly as efficient at inhibiting HIV-1 fusion and entry into targetcells as PA14 and 2D7, whose epitopes lie outside this region. PA14recognizes the tip of the Nt and residues in ECL2, whereas the 2D7epitope is located exclusively in ECL2. At the mechanism of action ofthese mAbs can only be speculated. It may be that their binding to thefirst few residues of ECL2 induces conformational changes in theco-receptor that prevent membrane fusion. Alternatively, obstruction ofECL2 epitopes might impede co-receptor oligomerization and the formationof a fusion-competent protein complex. Yet another possibility is thatresidues in ECL2 face the inside of the fusion pore and binding of themAbs impedes gp41 from inserting the fusion peptide into the plasmamembrane. In contrast, mAbs PA8-PA12 probably inhibit fusion and entryonly by directly competing for binding with gp120/CD4 complexes. We donot know if parameters other than epitope exposure and affinity for CCR5determine the efficacy of viral entry inhibition by these mAbs. It isunclear why inhibiting steps subsequent to the gp120/co-receptorinteraction would be more efficient than directly blocking thatinteraction. One way to explain this would be to assume that the offrate of gp120 binding to CCR5 is much lower than the on rate of mAbbinding to CCR5. Thus, every time a mAb detaches itself from aco-receptor molecule, a virion-associated gp120 molecule replaces it ina quasi-irreversible fashion since this interaction leads to membranefusion.

Synergy between combinations of anti-CCR5 mAbs is probably a result oftheir interactions with distinct epitopes that are involved ininter-dependent, consecutive steps of HIV-1 entry. The degree of synergyobserved between PA12 and 2D7 (CI<0.1 under many circumstances) isextraordinary since CI values <0.2 are rarely observed for combinationsof anti-HIV-1 antibodies (33, 35, 61), reverse transcriptase inhibitors(29), or protease inhibitors (44). Because of its potency, the PA12:2 D7combination was examined in multiple assay formats and concentrationratios, for which consistently high levels of synergy were observed.Moderate synergy was observed for PA12 combined with PA14. We alsoobserved moderate synergy between PA12 and CD4-IgG2. The CD4/gp120complex is metastable and if it is unable to interact with aco-receptor, decays into a non-fusogenic state (45-48). Since PA12directly blocks the gp120-binding site on CCR5, its presence may shiftthe equilibrium towards inactivation of the gp120/CD4 complex. Thiswould explain why we observe synergy between CD4-IgG2 and mAb PA12 withrespect to inhibition of fusion and entry. The lack of synergy betweenmAb PA14 and CD4-IgG2 suggests that they act on two non-consecutive andindependent steps of viral entry. A combination of further studies willbe needed to determine the precise mechanisms of synergy of thedifferent compounds with respect to inhibition of viral fusion andentry.

The above results are consistent with a model wherein HIV-1 entry occursin three distinct steps involving receptor binding, co-receptor binding,and co-receptor mediated membrane fusion. Separate co-receptor bindingand fusion events are suggested by the lack of correlation between themonoclonal antibodies' abilities to block gp120 binding and HIV-1fusion/entry. The chronology of events during fusion is furthersuggested by the patterns of synergies observed. Agents, such as PA12,that potently inhibit the middle step of the process, namely gp120binding, act synergistically with inhibitors of prior and subsequentsteps.

Example 2

Background: The increasing incidence of multidrug-resistant HIV-1mandates the search for novel classes of antiretroviral agents. CCR5 isa requisite fusion coreceptor for primary HIV-1 isolates and provides apromising target for antiviral therapy. PRO140 is an anti-CCR5monoclonal antibody that potently inhibits HIV-1 entry and replicationat concentrations that do not affect CCR5's chemokine receptor activityin vitro. In the present study, we evaluated the therapeutic potentialof PRO 140 in vivo using a therapeutic animal model of HIV-1 infection.

Methods: CD-17 SCID mice were reconstituted with normal human PBMC andinfected with the R5 isolate HIV-1 JR-CSF. When viral steady state wasreached, the animal were treated intraperitoneally with PRO 140 orcontrol antibody and monitored for viral burden using the Roche Amplicorassay. Initial studies examined a single 1 mg dose of PRO140. Inmulti-dose studies, PRO 140 was administered once every three days forthree weeks at doses ranging from 0.1-1.0 mg. In a separate experiment,flow cytometry was used to examine the potential for lymphocytedepletion following PRO 140 injection.

Results: Both single-dose and multi-dose PRO 140 reduced viral loads toundetectable levels in all treated animals, and the viral loadreductions ranged to 1.8 log 10. A transitory control of viralreplication was observed following single injection of PRO 140 whilemultiple injections led to a prolonged control with no evidence of viralrebound during therapy. Dose-dependent differences were observed in thekinetics of the PRO 140-mediated reductions in viral load. Flowcytometry analysis showed that treatment with PRO 140 did not lead tolymphocyte depletion, confirming that impact on viral replication invivo was solely due to CCR5-blockage.

Conclusions: PRO 140 is highly effective in controlling establishedHIV-1 infection in the hu-PBL-SCID mouse model of HIV-1 infection. Thesefindings provide in vivo proof-of-concept for PRO 140 therapy inparticular and for CCR5-inhibitors therapy in general.

Example 3 Methods

A humanized CCR5 antibody (huPRO 140) was tested for the ability toblock RANTES-induced calcium mobilization in L1.2-CCR5 cells and theability to block replication of HIV-1 CASE C 1/85 in human PBMC's usingmethods described herein.

Results:

The results as shown in FIG. 19 shows that the humanized CCR5 antibodypotently blocks HIV-1 but not RANTES.

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1-73. (canceled)
 74. An anti-CCR5 antibody fragment comprising anantibody fragment selected from the group consisting of: (a) a lightchain, which light chain comprises the expression product of a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097); (b) aheavy chain, which heavy chain comprises the expression product ofeither a plasmid designated pVg4:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg4:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099); (c) a Fab fragment whichcomprises (i) two light chains, each light chain comprising theexpression product of a plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097), and (ii) two heavy chains, each heavy chaincomprising the expression product of either a plasmid designatedpVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099); and (d) a F(ab′)₂ fragment which comprises (i) two lightchains, each light chain comprising the expression product of a plasmiddesignated pVK:HuPRO140-VK (ATCC Deposit Designation PTA-4097), and (ii)two heavy chains, each heavy chain comprising the expression product ofeither a plasmid designated pVg4:HuPRO140 HG2-VH (ATCC DepositDesignation PTA-4098) or a plasmid designated pVg4:HuPRO140 (mutB+D+I)-VH (ATCC Deposit Designation PTA-4099); and which antibodyfragment binds to CCR5 on the surface of a human cell.
 75. The anti-CCR5antibody fragment of claim 74, wherein the antibody fragment is thelight chain expressed by the plasmid designated pVK:HuPRO140-VK (ATCCDeposit Designation PTA-4097).
 76. The anti-CCR5 antibody fragment ofclaim 74, wherein the antibody fragment is the heavy chain expressed bythe plasmid designated pVg4:HuPRO140 HG2-VH (ATCC Deposit DesignationPTA-4098).
 77. The anti-CCR5 antibody of claim 74, wherein the antibodyfragment is the heavy chain expressed by the plasmid designatedpVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099). 78.The anti-CCR5 antibody fragment of claim 74, wherein the antibodyfragment is the Fab fragment of the anti-CCR5 antibody which comprises(i) two light chains, each light chain comprising the expression productof a plasmid designated pVK:HuPRO140-VK (ATCC Deposit DesignationPTA-4097), and (ii) two heavy chains, each heavy chain comprising theexpression product of either a plasmid designated pVg4:HuPRO140 HG2-VH(ATCC Deposit Designation PTA-4098) or a plasmid designatedpVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit Designation PTA-4099). 79.The anti-CCR5 antibody fragment of claim 74, wherein the antibodyfragment is the F(ab′)₂ fragment of the anti-CCR5 antibody whichcomprises (i) two light chains, each light chain comprising theexpression product of a plasmid designated pVK:HuPRO140-VK (ATCC DepositDesignation PTA-4097), and (ii) two heavy chains, each heavy chaincomprising the expression product of either a plasmid designatedpVg4:HuPRO140 HG2-VH (ATCC Deposit Designation PTA-4098) or a plasmiddesignated pVg4:HuPRO140 (mut B+D+I)-VH (ATCC Deposit DesignationPTA-4099).
 80. An anti-CCR5 antibody fragment comprising (i) a lightchain which comprises consecutive amino acids having the sequence setforth in SEQ ID NO:6; or (ii) a heavy chain which comprises consecutiveamino acids having the sequence set forth in SEQ ID NO:9 or SEQ IDNO:12.
 81. A composition comprising the anti-CCR5 antibody fragment ofclaim 74 or claim 80 and a carrier, a diluent or an excipient.
 82. Thecomposition of claim 81, wherein the anti-CCR5 antibody fragment hasattached thereto a material selected from the group consisting of aradioisotope, a toxin, polyethylene glycol, a cytotoxic agent and adetectable label.
 83. A method of inhibiting HIV-1 infection of a CD4+cell which comprises contacting the CD4+ cell with the anti-CCR5antibody fragment of claim 74 or claim 80, in an amount and underconditions such that fusion of HIV-1 or an HIV-1 infected cell to theCD4+ cell is inhibited, thereby inhibiting HIV-1 infection of the CD4+cell.
 84. The method of claim 83, which further comprises labeling theanti-CCR5 antibody fragment with a detectable marker.
 85. The method ofclaim 84, wherein the detectable marker is a radioactive or afluorescent marker.
 86. The method of claim 83, wherein the CD4+ cellexpresses CCR5.
 87. A method of treating a subject afflicted with HIV-1which comprises administering to the subject an effective HIV-1 treatingdosage amount of the composition of claim 81, under conditions effectiveto treat said HIV-1-afflicted subject.
 88. The method of claim 87,wherein the composition is administered to the subject by a methodselected from the group consisting of intravenous, intramuscular andsubcutaneous means.
 89. The method of claim 87, wherein the compositionis administered continuously to said subject or at predeterminedperiodic intervals.
 90. The method of claim 87, wherein the dosage ofsaid composition ranges from about 0.1 to about 100,000 μg/kg bodyweight of said subject.
 91. The method of claim 87, wherein the dosageof said composition does not inhibit an endogenous chemokine activity onCCR5 in said subject.
 92. A method of preventing a subject fromcontracting an HIV-1 infection which comprises administering to thesubject an effective HIV-1 infection-preventing dosage amount of thecomposition of claim 81, under conditions effective to prevent saidHIV-1 infection in said subject.
 93. The method of claim 92, wherein theanti-CCR5 antibody fragment is administered to the subject by a methodselected from the group consisting of intravenous, intramuscular andsubcutaneous means.
 94. The method of claim 92, wherein the anti-CCR5antibody fragment is administered continuously to said subject or atpredetermined periodic intervals.
 95. The method of claim 92, whereinthe dosage of said anti-CCR5 antibody fragment ranges from about 0.1 toabout 100,000 μg/kg body weight of said subject.
 96. The method of claim92, wherein the dosage of said anti-CCR5 antibody fragment does notinhibit an endogenous chemokine activity on CCR5 in said subject.